U.S. patent application number 10/638723 was filed with the patent office on 2004-12-09 for recognition molecules interacting specifically with the active site or cleft of a target molecule.
Invention is credited to Muyldermans, Serge, Wyns, Lode.
Application Number | 20040248201 10/638723 |
Document ID | / |
Family ID | 33492172 |
Filed Date | 2004-12-09 |
United States Patent
Application |
20040248201 |
Kind Code |
A1 |
Muyldermans, Serge ; et
al. |
December 9, 2004 |
Recognition molecules interacting specifically with the active site
or cleft of a target molecule
Abstract
The invention relates to a recognition molecule capable of
interacting with an active site or cleft of a target molecule. The
recognition molecule includes an exposed loop structure that
extends from a basic recognition unit. The loop structure is, for
example, the CDR3 of a camelid species heavy chain antibody having
a binding specificity for the active site or cleft of a target
molecule, or a derived version of such a CDR3. The basic
recognition unit can be formed by an antibody-type structure having
binding affinity for the target molecule.
Inventors: |
Muyldermans, Serge;
(Hoeilaart, BE) ; Wyns, Lode; (Antwerpen,
BE) |
Correspondence
Address: |
WEBB ZIESENHEIM LOGSDON ORKIN & HANSON, P.C.
700 KOPPERS BUILDING
436 SEVENTH AVENUE
PITTSBURGH
PA
15219
US
|
Family ID: |
33492172 |
Appl. No.: |
10/638723 |
Filed: |
August 11, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10638723 |
Aug 11, 2003 |
|
|
|
09214027 |
Oct 25, 1999 |
|
|
|
09214027 |
Oct 25, 1999 |
|
|
|
PCT/EP97/03488 |
Jun 27, 1997 |
|
|
|
Current U.S.
Class: |
435/7.1 ;
436/518; 506/18; 506/9; 530/388.22 |
Current CPC
Class: |
C07K 16/40 20130101;
A61K 38/00 20130101; C07K 16/12 20130101; C07K 16/18 20130101; C07K
16/00 20130101; A61K 39/00 20130101 |
Class at
Publication: |
435/007.1 ;
436/518; 530/388.22 |
International
Class: |
G01N 033/53; G01N
033/543; C07K 016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 27, 1996 |
EP |
96201788.5 |
Claims
1-21. (cancelled).
22. A method for isolating a sequence encoding a polypeptide which
binds to the active site of a target molecule, comprising: (a)
providing a library of coding sequences of camelid heavy chain
antibodies or camelized heavy chain antibodies; (b) expressing the
coding sequences of the library of camelid heavy chain antibodies
or camelized heavy chain antibodies; (c) selecting among the
expressed coding sequences a camelid heavy chain antibody or
camelized heavy chain antibody which binds to the active site of
the target molecule; and (d) isolating the sequence encoding the
camelid heavy chain antibody or camelized heavy chain antibody
selected at step (c), wherein the active site is selected from the
group consisting of a catalytic site of a protein having enzymatic
activity, a toxic site of a toxin, a toxic site of a venom, and a
recognition site of a receptor.
23. The method of claim 22, further comprising: (a) providing a
library of coding sequences of camelid heavy chain antibodies or
camelized heavy chain antibodies in phage display vectors; (b)
expressing the coding sequences on phages harboring phage display
vectors; (c) selecting the phage expressing the polypeptide which
binds to the active site of the target molecule by panning the
phage library with the immobilized target molecule; and (d)
isolating the sequence encoding the polypeptide which binds to the
active site of the target molecule from the phage selected at step
(c).
24. The method of claim 22, further comprising providing the
library of coding sequences of camelid heavy chain antibodies or
camelized heavy chain antibodies by: (a) immunizing a camel with
the target molecule; (b) isolating the coding sequences of camelid
heavy chain antibodies or camelized heavy chain antibodies from the
lymphocytes of the immunized camel; and (c) cloning the isolated
coding sequences into vectors, wherein a library of coding
sequences of camelid heavy chain antibodies or camelized heavy
chain antibodies is provided.
25. The method of claim 22, further comprising providing the
library of coding sequences of camelid heavy chain antibodies or
camelized heavy chain antibodies by: (a) providing a random camelid
heavy chain antibody or a random camelized heavy chain antibody;
(b) isolating and cloning the coding sequence of the random camelid
heavy chain antibody or camelized heavy chain antibody; (c)
modifying the coding sequence of the random camelid or camelized
heavy chain antibody in the CDR3 region by random mutagenesis of at
least one of the codons thereof; and (d) preparing the library
consisting of the randomly mutated coding sequences.
26. The method of claim 25, wherein the coding sequence of the
random camelid heavy chain antibody or camelized heavy chain
antibody in the CDR3 region is modified by exchange of at least one
codon by one or more random condons.
27. The method of claim 26, wherein at least the codons 97-102 are
replaced by random codons varying from 1 to 10 codons.
28. The method of claim 25, further comprising modifying the coding
sequence of the random camelid heavy chain antibody or camelized
heavy chain antibody to generate amino acid substitutions in the
CDR1 or CDR2 regions in order to increase the specificity and/or
the affinity of the polypeptide to the active site of the target
molecule.
29. The method of claim 23, wherein the step of selecting the
phages expressing the polypeptide which binds to the active site of
the target molecule by panning the phage library with the
immobilized target molecule further comprises eluting the binding
polypeptides with an excess of substrates or inhibitors.
30. The method of claim 22, wherein the target molecule is a
bacterial toxin selected from the group consisting of toxins
secreted by S. aureus, staphylococcal enterotoxin B proteins,
cholera toxins, and tetanus toxin.
31. The method of claim 22, wherein the target molecule is a snake
venom selected from the group consisting of adamalysin II,
cardiotoxin CTX lib, cardiotoxin CTX V, dendrotoxin K, flavoridin
neurotoxin-I and II, and metalloproteinase Ht-c and Ht-d.
32. The method of claim 22, wherein the target molecule is selected
from the group consisting of HIV protease, HIV reverse
transcriptase, SIV protease, alkaline protease from Pseudomonas
aeruginosa, serine proteases, RNAses, angiogenin, sialidases,
amylases, and .beta.-glucanases.
33. The method of claim 22, wherein the target molecule is a
therapeutic or a diagnostic target.
34. The method of claim 22 wherein the target molecule is a
receptor.
35. The method of claim 23, wherein the isolated sequence is a
chemically synthesized analogue or a peptidomimetic.
36. The method of claim 23, wherein the step of selecting the
phages expressing the polypeptide which binds to the active site of
the target molecule by panning the phage library with the
immobilized target molecule further comprises eluting the binding
polypeptide using an excess of receptor agonist or antagonist.
Description
[0001] The present invention relates to recognition molecules,
which are capable of interacting with the active site or cleft of
target molecules. The invention further relates to methods for
their design and preparation and use of the recognition molecules
in diagnosis, therapy, vaccines and methods for isolation or
purification of target molecules. Preferably the recognition
molecule are used as enzyme inhibitors. The invention also relates
to therapeutical compositions, diagnostic kits, vaccines and
purification materials, comprising the recognition molecules of the
invention.
[0002] In theory, in many instances the outbreak of diseases from
viral, bacterial, parasitic or any other origin can be avoided by
interfering with the enzymatic activity of pathogenic proteins or
with the recognition of parasitic proteins with their target
molecules. Furthermore, the deleterious effect of toxic substances
can be counteracted by binding inhibiting molecules at the active
(toxic) site. Also the malfunction of complex enzymatic or
physiological processes finding their origin in a deregulated
enzymatic function or deregulated protein recognition, often can be
cured by adding molecules interacting with the active site or
grooves of the complex proteins.
[0003] In all these examples it would be advantageous to have
specific proteins recognizing the active site (such as the
catalytic site in enzymes, grooves in proteins of complex systems,
such as multicomponent systems, or recognition sites in receptors)
of these malignant molecules.
[0004] Obviously the best technique at hand nowadays to obtain such
molecules recognizing a particular target molecule is hybridoma
technology for generating (monoclonal) antibodies. However,
antibodies impose several limitations on their exploitation. It is
for example known that antibodies of which the structure has been
solved up to now, have an antigen binding surface forming either a
groove or cavity itself or a flat surface (Webster et al., Current
Opinion in Structural Biology, 4, 123, 1994). Thus, the antigen
binding site of the antibodies cannot penetrate a groove or cavity.
The catalytic or functional residues or toxic parts of the target
proteins are located mostly within a cleft, so that the recognition
of their substrate or receptor becomes very specific due to the
many contacts and interactions with the amino acids forming the
active cleft. However, due to the fact that clefts and cavities lie
at least partially within the molecule, these structures are not
very immunogenic. Even the wider cavities--or clefts of proteins in
general--have the disadvantage that they are not very immunogenic.
This is one of the main reasons why so few antibodies are
interacting with the active site of proteins.
[0005] These (the low immunogenicity of the active site and the
flat surface of the antigen binding site itself) are probably the
two main reasons why so few (monoclonal) antibodies appear to have
neutralizing enzymatic activity certainly when acting as monovalent
fragments (F.sub.AB-F.sub.v-ScF.sub.v), and this puts severe
limitations on the great potential of monoclonal antibodies. The
few neutralizing monoclonal antibodies that are available appear to
bind on epitopes which overlap partially the active site of the
antigen, but not inside the active site which would give them a
greater specificity. Furthermore, a simple point mutation at the
surface of the antigen (such as viral coat protein) removes the
epitope of the monoclonal antibody which becomes useless for
detecting this new variant. A last disadvantage of antibodies is
their large molecular weight and size which impose limitations on
the fast bio-distribution or efficient tissue penetration, while
the Fc of antibodies prevents a fast clearance from blood.
[0006] In view of the above it is a first object of the present
invention to design and construct molecules which bind with a great
specificity to a cavity or active site of a target molecule.
[0007] It is a further object of the present invention to use these
new molecules, which will be designated `recognition molecules`
throughout this specification, in diagnosis, therapy, vaccines and
methods for isolating or purifying target molecules and as enzyme
inhibitors.
[0008] The third object of the invention is to provide for methods
for preparing and modifying the recognition molecules for specific
purposes.
[0009] According to a fourth object the invention relates to
therapeutical compositions, diagnostic kits, vaccines and
purification materials, comprising the recognition molecules of the
invention.
[0010] The first object of the invention is achieved by a
recognition molecule, being capable of interacting with an active
site or cleft of a target molecule, which recognition molecule
comprises an exposed loop structure, which extends from a basic
recognition unit.
[0011] In such a recognition molecule the loop structure is
preferably the Complementary-Determining Region 3 (CDR3) of a
camelid species heavy chain antibody having a binding specificity
for the active site or cleft of the target molecule, or a modified
version thereof. The loop can be incorporated in any available
basic recognition unit. However, preferably the basic recognition
unit is formed by an antibody-type structure having also at least
some binding affinity for the target molecule.
[0012] The invention was made after first having formulated the
following principles. The recognition molecule of the invention
should consist of an exposed loop protruding from a recognition
unit (FIG. 1). The loop should be designed to penetrate inside a
cavity, groove or cleft of the target protein. To have a good
affinity this loop needs to be constrained so that its inherent
flexibility is limited. Therefore the flexibility of the free loop
needs to be restricted in absence of the target molecule. Although
a restricted loop on itself might already have a sufficient
affinity, it is an advantage (but not critically required) to have
this loop protruding from a binding-surface to increase the number
of contacts with the amino acids around the active site or cleft of
the target protein. Thus, the ideal recognition molecule is
something like the antigen-binding site of an antibody topped with
an exposed loop protruding from this antigen-binding surface.
[0013] However, it was considered by the present inventors that the
antigen binding site of conventional antibodies or antibody
fragments such as Fv is not a good starting scaffold for insertion
of an exposed loop because antibodies normally do not form loops
protruding from their antigenic binding site and an artificially
created loop is difficult to design de novo as the loop needs to be
structured, constrained and should possess a complementary surface
to the cavity of the target.
[0014] Moreover antibodies are too large for an efficient
bio-distribution or tissue penetration, and antibody fragments such
as Fv are rather unstable and easy to dissociate especially when
used at lower concentrations.
[0015] Also, the much smaller VH antibody fragments or `single
domain antibody` (dAb) as they were called, (Ward et al., Nature
341, 544-546, 1989) derived from a conventional antibody have three
major limitations, namely low expression yield in bacteria (only
0.2 mg/l culture on average), low solubility in aqueous solution,
and reduced affinity and specificity compared to the parental Fv
fragment.
[0016] The molecule of the present invention should preferably
penetrate into the active site of the target protein, where it
interacts with the catalytic residues, although other cavities,
grooves or clefts of the target protein will do as well. To this
end the molecule of the invention should possess an exposed loop,
large enough to insert maximally, and as complementary as possible
to the target protein cavity. The good fit, and high number of
contacts of the recognition molecule with the active site residues
of the target molecule should make it impossible for the latter to
escape this interaction by the acquisition of point mutations as
these will have deleterious effect on the proper function of the
target molecule itself. The recognition molecule of the invention
is further to be characterized by a small size for good
bio-distribution and tissue penetration, sufficient high expression
level in bacterial systems for economical reasons, a good
solubility behaviour, a good stability and a long shelf-life time,
a good affinity and specificity for the target molecule, an easy
cloning and downstream manipulation.
[0017] The present inventors surprisingly found that all these
requirements can be met when starting out from the heavy chain
antibodies from camelids (Camel bactrianus, Camel dromedarius, Lama
peruviana, Lama glama, Lama vicuana and Lama alpaca). Camelids
contain a substantial amount of their functional antibodies in the
form of heavy-chain antibodies only (Hamers-Casterman et al.,
Nature 363, 446, 1993). The heavy-chain antibody is composed of
homodimers of H chains and lack L chains. From the amino acid
sequence of the H chains it appears that their N-terminal domain
harbours some remarkable amino acid substitutions which make them
clearly distinct from the conventional VH (Muyldermans et al.,
Prot. Enging. 7, 1129, 1994).
[0018] To make a clear distinction between the conventional VH and
that of camelids, the heavy-chain antibody of the latter is
identified as `VHH` (VH of Heavy-chain antibody). This distinction
is entirely justified as follows from the fact that camelid
heavy-chain antibodies are clearly different from any other VH.
[0019] The fact that the camelid germline contains both a V.sub.H
and a V.sub.HH set of minigenes proves that the V.sub.HH domains
(obtained after V.sub.HH-D-J-recombination) are predestinated for
usage in heavy-chain antibodies devoid of light chains.
[0020] Although the amino acid substitutions in a VHH compared to
any other VH are scattered throughout the primary structure
(sequence) they are clustered in space in the tertiary structure at
the side of the molecule which normally interacts with the VL
domain (referred to as `former VL side`). These amino acid
substitutions are V37F, G44E, L45R or L45C and W47 mostly
substituted in Gly. Evidently, such substitutions are expected to
render the `former VL side` of the VHH more hydrophilic and
therefore they will overcome the solubility limitations of the
conventional isolated VH's obtained from human or mouse also
referred to as dAb (single domain antibodies). Ward et al., Nature
341, 544 (1989) described the so-called dab's.
[0021] The substitutions also make that this region is less likely
to bind to the chaperon BiP (or bacterial chaperon proteins) so
that it is expected that the expression level might also
increase.
[0022] Moreover, as the camelid heavy chain antibodies were matured
in vivo in absence of any light chain, it was anticipated that the
isolated VHH will retain the parental affinity and specificity for
its antigen of the original heavy chain antibody.
[0023] In conclusion, VHH's have at least three main advantages
compared to the isolated VH's or dAb's, namely better expression
yield in bacteria or other expression systems, a better solubility
in aqueous solutions and an increased affinity and specificity.
[0024] Indeed, it was demonstrated in the research that led to the
present invention that a VHH when cloned in a bacterial expression
vector (pHEN4, a derivative of the pHEN1 (described by Hoogenboom
et al., Nucl. Acids Res. 19, 4133 (1991))) can be expressed to
yield approximately 10 mg/l culture. This should be compared to 0.2
mgr/l culture for bacterial expression of isolated mouse VH
domains. The cause of these unfavorable properties of mouse dAb's
is the exposure to aqueous solvent of the hydrophobic face of
`former VL side`. The VHH could also easily be concentrated to 10
mg/ml without any sign of aggregation, which corresponds to an
approximately a 100 times higher solubility than that of mouse
VH's.
[0025] Besides the good bacterial expression and solubility it was
further shown that the VHH was resistant against thermal
denaturation and could be kept at 37.degree. C. for up to 1 week
with retention of structural integrity and antigen binding
capacity. It was therefore concluded that the VHH are stable
molecules.
[0026] It is possible to camelise ordinary VH's to equip them with
the advantages of VHH. The so-called `camelisation` involves the
mutagenesis of amino acids at position 44, 45 and 47 so as to mimic
the corresponding camel amino acids at those positions.
`Camelisation` improves on the solubility (Davies & Reichmann
FEBS Lett. 339, 285-290, 1994). Further `camelisation` of position
37 by V37F substitution and the introduction of a disulphide bond
between the CDR1 and CDR3 improved considerably the stability of
the isolated domain.
[0027] The sequence analysis of additional VHH clones (from camel
and llama) revealed some remarkable additional features about their
functionality, especially how they retained a specific antigen
binding capacity in the absence of any light chain antigen binding
loops. In a conventional VH, it is the CDR1 (amino acid 31 to 35)
which is hypervariable in sequence and found to contact the
antigen. The N-end in front of the first hypervariable loop (amino
acid 26 to 30) is solvent exposed in a conventional VH, however,
these amino acids are conserved and were never before reported to
contact the antigen (with the exception of amino acid at position
30).
[0028] In the VHH of camelids these amino acids at position 26 to
35 can be defined as hypervariable in sequence. This suggests,
first, that the amino acids of this loop can adopt a different
conformation than that described up to now in other species, and
second, it indicates that those amino acids might contact the
antigen, i.e. the surface of the antigen binding platform would be
enlarged.
[0029] Furthermore, it was found that the CDR3 which is the most
variable loop in sequence and in structure is on average longer
than that of conventional VH domains (15 amino acids compared to 9
amino acids in mice). Again an increase in the antigen binding
surface is anticipated.
[0030] A drawback of a longer loop in absence of the antigen means
that the loop has some flexibility and may adopt more different
conformations of which one becomes fixed upon complexation with the
antigen. This immobilisation of the loop will have a large negative
entropic effect on binding. The frequent occurrence (especially in
the longer loops) of cysteines simultaneously in the CDR1 (or CDR2
or position 45 of framework 2) and the CDR3 of camelid VHHs is in
accordance with the formation of a disulphide bond. This will
reduce the conformational flexibility and therefore the antigen
binding will have less negative entropic contribution in
binding.
[0031] From the results of VHH and the `camelised VH` two
independent strategies can be proposed for generating functional
small recognition units.
[0032] The first strategy comprises taking phage displayed
`camelised VH` as a scaffold and use this to randomise the CDR3
loop (CDR3 to start with; in a subsequent step eventually the CDR1
and CDR2 loops can be randomised/mutated) to fine-tune the affinity
and specificity.
[0033] The second strategy comprises immunising a camel (or llama)
with the desired antigen so that the immune system of the animal
will mature his heavy-chain antibodies in vivo. Subsequently the
VHH from the lymphocytes (blood, spleen, bone marrow) are cloned in
a phage display vector such as the pHEN4, and selected by panning
with the antigen. Using this technique successful identification of
two VHH molecules binding to different epitopes of lysozyme and two
VHH binders binding to tetanus toxoid, one to the C-fragment and
the other outside the C-fragment was possible. These VHH were
called cAb-Lys2, cAb-Lys3 and cAB-TT1 and cAb-TT2, respectively.
See FIG. 2 for their amino acid and nucleotide sequences with Kabat
numbering. (cAb stands for camel single domain antibody
fragment.)
[0034] The cAb's are specific for their antigen, and bind to it
with affinities of 2.10.sup.8, 2.10.sup.7, 6.10.sup.7 and
2.10.sup.7 M.sup.-1 respectively. The bacterial expression levels
of these cAb's are always in the mg/l culture range, the cAb's are
well folded and behave quite soluble and stable in thermal
denaturation experiments.
[0035] It is possible to increase their affinity by making the
cAb's bivalent/multivalent by the intermediate of the camel long
hinge. In a similar strategy the cAb-Lys3 can be linked to the
cAb-TT2 to generate bispecific constructs. The cAb-TT1 and cAb-TT2
are shown to neutralise the tetanus toxin in vivo. The cAb-Lys3 is
inhibiting the Micrococcus Lysodeikticus cell wall hydrolysing
activity of lysozyme as well.
[0036] The cAb-Lys3 is readily purified by affinity chromatography
on hen egg-white lysozyme immobilised on Sepharose CNBr. The
structure of the cAb-Lys3 in complex with its antigen (hen
egg-white lysozyme) was determined to 2.5 .ANG. resolution by X-ray
crystallography. The main observations of the cAb-Lys3 structure
with respect to the development or design of VHH as small
recognition units with the exposed loop structure (also called
`TUT` motif) are the following.
[0037] The main chain conformation of the core of the VHH is
similar to the VH, so that it can be envisaged to use the VHH for
grafting the CDR's from other useful conventional VH molecules.
[0038] The `former VL` side of the cAb-Lys3 is completely reshaped
and became more hydrophilic compared to the VH, due to the
substitution of the V37F, G44E, L45R and W47 mostly substituted in
Gly, but also due to the reorientation of the conserved W103, Q105
and Q39 in this region.
[0039] The H1 loop has a conformation which deviates completely
from the described canonical structure 1 of conventional VH's, and
amino acid 29 which in conventional VH domains is buried in the
interior of the domain flips out the structure and contacts the
antigen. Also the amino acid 28 is close to the antigen in the
complex.
[0040] The CDR3 (24 amino acids long in cAb-Lys3) can be divided
into two parts, the C-part covering the `former VL side`, and the
N-part forming an exposed, accessible, extended and protruding loop
(referred to as the `TUT`). This loop is stabilised by a disulphide
bond (towards the CDR1) and an internal aromatic core formed by a
clustering of Y32, Y99 and Y100c. The Y99 is also making a H-bond
with the side chain of D95.
[0041] The loop at amino acids 72-75 is close to the antigen, but
is not very well ordered.
[0042] The exposed part of the CDR3 loop penetrates deeply inside
the active site of the lysozyme, and the tip of the loop formed by
Ala100 and Ser100a. The Ser100a makes a H-bond with the catalytic
Glu35 of lysozyme.
[0043] It was found that the stabilised and large protruding loop
(the `TUT`) interacts with the active cleft of the lysozyme, an
enzyme region considered to be a `low energetic epitope`, because
of which it is difficult to raise antibodies against this part of
the molecule. The active site of the enzyme, or cavities of protein
surface in general, are difficult to react with conventional
antibodies or antibody fragments due to their low immunogenicity or
because the antigen binding site is either flat or carries a cavity
or groove, but a large protruding loop was not observed on the
antigen binding site of antibodies so that they cannot penetrate
cavities, clefts or grooves of the antigen.
[0044] The recognition molecules of the invention are in particular
peptide-like structures. A broad range of proteins or other
molecules can function as target molecules. A list of proteins is
given merely as an indication of which kind of proteins can be
selected. All other proteins with a cavity or small groove are as
good, and the list is certainly not limited.
[0045] Examples of target molecules are bacterial toxins, such as
Toxic Shock Syndrome Toxin 1 of S.aureus, which is a member of a
large family of toxins secreted by S.aureus and is the major cause
of toxic shock syndrome. TSST-1 has 20-30% sequence identity with
staphylococcal enterotoxin B proteins, cholera toxin, tetanus
toxin. Other molecules that may be selected as target molecules are
snake venoms, such as adamalysin II, a smaller protein, which is a
zinc endopeptidase from rattlesnake and consists of a highly
conserved catalytic domain, or Cardiotoxin CTX IIb (Naja
mosambica), Cardiotoxin CTX V (Taiwan Cobra). These cardiotoxins
are small proteins in the venoms of snakes from the Elapidae
family. The toxins are known to bind and disrupt the organization,
integrity and function of the cell membrane. Others are Dendrotoxin
K (Black mamba), Flavoridin Neurotoxin-I and II (Asian cobra).
There is also a sequence similarity of adamalysin II to the low
molecular weight metalloproteinase Ht-c and Ht-d from the Crotalus
atrox which degrades type IV collagen.
[0046] Other target molecules are for example receptors. Receptors
are biological macromolecules capable to bind, a complementary
biomolecule, or counterligand, resulting in a function (through,
e.g., signal transduction or storage and subsequent release). The
recognition molecules of the invention can be used as antagonists
to receptors in order to block for example the signal transducing
function thereof. As an alternative the recognition molecules can
have a agonistic activity.
[0047] Target molecules can furthermore be honey bee venoms, such
as apamin and tertiapin, spider toxins, viral and bacterial
specific proteins, such as HIV protease, HIV reverse transcriptase,
SIV protease, alkaline protease from Pseudomonas aeruginosa, other
proteases, such as serine proteases like Factor Xa or other blood
serine proteases, RNases and angiogenin, sialidases thought to be
involved in the pathogenesis of many diseases (Salmonella,
influenza virus), which catalyse the cleavage of glycosidic
linkages between sialic acid and glycoconjugates, amylases and
.beta.-glucanases, which catalyse the hydrolysis of glycosidic
linkages of various oligosaccharides. Changes in .alpha.-amylase
activity are often indicative of pancreatic disorders. The active
site of the enzyme forms a cleft that lies between the A and B
domains. The catalytic residues Asp300, Asp197 and Glu233 are
located at the cleft and homologous residues have been found within
several amylase structures.
[0048] Other examples of target molecules are lysozyme, tetanus
toxin and carbonic anhydrase.
[0049] Starting from a basic recognition molecule, consisting of a
basic recognition unit and a loop structure variations can be made
to make recognition molecules for other targets. Furthermore, a
selection system can be designed to select suitable candidates for
a desired target from within a large group of variants.
[0050] In principle every recognition molecule can be used as a
starting point for this approach. However, to avoid further
immunization use can be made from one of the recognition molecules
described herein. Such a molecule can then be engineered to obtain
the desired specificity.
[0051] Variations leading to modified versions of the loop or the
basic recognition unit can be made in various ways. For example,
the derived version of the CDR3 may be a mutated CDR3 in which at
least one of its native amino acids is replaced by one or more
other amino acids. Or as an alternative the derived version of the
CDR3 consists of a mutated CDR3 in which one or more additional
amino acids are added to and/or incorporated within its native
amino acid sequence.
[0052] Similar modifications can be made to the basic recognition
unit. For example, when the basic recognition unit is an
antibody-type structure formed by at least part of a camelid
species heavy chain antibody, a modified version thereof is a
version in which at least one of its native amino acids is replaced
by one or more other amino acids, or a version in which one or more
additional amino acids are added to and/or incorporated within its
native amino acid sequence. As an alternative the modified version
of the camelid species may comprise a version which is fused to a
second amino acid sequence or a biologically active molecule.
[0053] In order to obtain recognition molecules of the invention
various strategies can be followed. In general a first method
comprises providing a camelid heavy chain antibody; isolating and
cloning the coding sequence therefore in a phage display vector;
expressing the coding sequence on a phage harbouring the vector;
and selecting the recognition molecule specific for the antigen by
panning the phage with the immobilized antigen.
[0054] The first strategy thus consists of immunizing a camel (or
llama) with the desired target antigen so that the immune system of
the animal matures his heavy-chain antibodies in vivo against this
immunogen. Subsequently, the VHH's from the lymphocytes (blood,
spleen, bone marrow) are cloned in a phage display vector such as
the pHEN4, and selected by panning with the immobilized antigen. To
elute the binders (recognition molecules that bind the desired
antigen) one of the following methods was chosen.
[0055] The elution of binders is performed by a pH shock to obtain
general binders (not only active site binders). The elution of
binders is performed with an excess of substrate if one only wishes
to obtain the cavity or active site binders. By bringing the
`general binders` from the first method over an immobilized target
protein/substrate complex and continuing with the non-binders the
cavity binders are selected from the general binders.
[0056] The feasibility of this general method to obtain recognition
molecules of the invention was proved by obtaining the camel
single-domain antibody (cAb) fragments cAb-TT1, cAb-TT2, cAb-Lys2
and cAb-Lys3 proteins using this strategy. The structure analysis
of the cAb-Lys3, which has the longest CDR3 loop (24 amino acids)
of all lysozyme binders and a cysteine forming a disulfide bond
between the CDR1 and CDR3, proved that indeed a small recognition
unit with a protruding loop binding into the active site of the
enzyme was generated as anticipated.
[0057] It is possible to repeat the first strategy for obtaining
recognition molecules with other `target` proteins but not all
proteins are sufficiently immunogenic, to completely generalize
this strategy. Therefore, a second strategy was developed.
[0058] This strategy uses a randomly chosen recognition molecule as
a starting point. The advantage thereof is that immunization can be
avoided, and the tertiary structure of the final molecule is
already essentially provided for.
[0059] Therefore, such a second method comprises in general
selecting a random camelid heavy chain antibody; isolating and
cloning the coding sequence therefore in a phage display vector;
modifying the coding sequence of the exposed loop by random
substitution of at least one of the codons thereof; preparing a
library of randomly mutated coding sequences in phage display
vectors; expressing the coding sequence on phages harbouring the
vector; and selecting the recognition molecule specific for the
antigen by panning the phage with the immobilized antigen.
[0060] For this second strategy, the camel immunization is avoided.
For example, the cAb-Lys3 protein is modified to develop `small
recognition units` with a `TUT` motif for binding into the target
protein clefts. Two routes are envisaged: a first one in which the
protruding loop is reshaped and secondly a route ending in a
`veneering` of the cAb-Lys3 protruding loop.
[0061] To reshape the loop several steps need to be undertaken.
First, introduction of restriction enzyme sites in the vicinity of
the loop. These sites help in the subsequent cloning and
characterization. Then, exchange of the amino acids of the loop by
a random codons (1 to X). The smaller the number X, the smaller the
library, and the shorter the extension of the loop. Loops of more
than 6-7 amino acids might be more difficult to generate a complete
library due to the experimental limitations in bacterial
transformation efficiency, and also the loop might become too
flexible which will result in polyreactivity and make less tight
binders. Subsequently, exchange the platform around the loop by
changing the N terminal end of the domain, the CDR1, the CDR2 or
the loop around amino acids at position 72/75 (FIG. 2, cAb-Lys3) in
order to increase the specificity or affinity. The previous steps 2
and 3 can be repeated cyclically for affinity and specificity
maturation. Alternatively multivalent constructs can be made as
well to increase the avidity or to obtain bispecific
constructs.
[0062] For `veneering` the cAb-Lys3 CDR3 loop the following steps
are performed. First, introduction of restriction enzymes sites
around the loop for cloning or characterization purposes. Then,
substitution of the amino acids which are exposed on the outside of
the protruding loop of the cAb-Lys3. Finally, exchange of the
platform around the loop by changing the N terminal end, the CDR1,
the CDR2 or the amino acids around the 72/75 loop in order to
increase the specificity or affinity. Steps 2 and 3 might be
repeated for affinity and specificity maturation. Alternatively
multivalent constructs can be made as well to increase the avidity
or to obtain bispecific constructs.
[0063] It is also possible to produce the recognition molecules of
the invention by means of standard genetic engineering techniques,
such as expression of a DNA sequence encoding the recognition
molecule. Such a method may for example comprise the steps of
isolating a DNA sequence encoding the recognition molecule or a
precursor therefor; optionally modifying the molecule or the
precursor by introducing one or more base substitutions, deletions
or insertions; transferring the thus obtained optionally modified
DNA sequence to a suitable host; and expressing the DNA sequence in
the host. The term `precursor` as used herein intends to encompass
every sequence that does not have the (complete) desired
specificity of the recognition molecule to be produced.
[0064] It is evident that with these approaches the problems of
immunization (long immunization schemes, toxic effects of
immunogens to camelids, low immunogenicity of the target molecule,
need for relatively large amounts of target molecules for
immunization) are avoided.
[0065] According to another aspect thereof the invention relates to
use of the recognition molecules in neutralising the biological
function of the target molecule, and in therapy. The invention thus
also relates to a therapeutical composition, comprising one or more
recognition molecules of the invention and a suitable
excipient.
[0066] Apart from neutralising, the recognition molecules may also
be used to detect the presence of the target molecule in a sample.
As such the recognition molecules may be used for diagnosis. The
recognition molecules can be used analogous to conventional
antibodies. Similar diagnostic techniques are therefore envisaged
here, without further explanation, because the skilled person will
be very well capable of designing every conceivable diagnostic test
in analogy to known immunological diagnostic tests. The invention
thus provides for diagnostic test kits, comprising one or more
recognition molecules.
[0067] The recognition molecules, like conventional antibodies may
find application in (passive) vaccines. To this end, the invention
relates to a vaccine, comprising one or more recognition molecules
of the invention.
[0068] Furthermore, the specificity of the recognition molecule for
the target molecule can be deployed to isolate or further purify
the target molecule. Use can be made of standard separation and
purification techniques, such as affinity columns, wherein the
conventional antibody or other binding molecule is substituted with
the recognition molecule of the invention. The invention thus
further relates to purification material, consisting of a carrier
having one or more recognition molecules of the invention bound
thereto. Preferably, the carrier is column material preferably an
affinity column.
[0069] Once the small recognition unit with the `TUT` motif
(recognition molecule of the invention) is constructed, it can thus
be used immediately in a number of applications, for example
instead of a conventional monoclonal where a rapid clearance from
blood of excess molecule is advantageous. However, for other
applications it might be preferable to increase the lifetime in
circulating blood. This is readily obtained by cloning the
recognition unit in front of the hinge, CH2 and CH3 domains of
human IgG1.
[0070] In a third class of applications it might be necessary to
turn these small recognition units into intrabodies. The small
size, and their single domain architecture makes that they are
suitable for such use.
[0071] Cloning of a SKDEL motif at the end of the gene segment will
keep the molecule inside the endoplasmic reticulum, or cloning
behind a nuclear target signal, the chloroplast signal, will bring
the protein inside the nucleus, the chloroplast, the mitochondria
or any other selected organelle where it should bind and inactivate
its target.
[0072] For a number of cases, the binding activity of the loop on
itself might be sufficient for specific interaction inside the
active cleft of the target molecule.
[0073] The loop can also be used to rationally design
peptido-mimics preferably mimicking the properties of natural
proteins.
[0074] The present invention will be further elucidated referring
to the following figures and examples.
DESCRIPTION OF THE FIGURES
[0075] FIG. 1 shows a schematic representation of a recognition
molecule of the invention.
[0076] FIG. 2 shows the amino acid and nucleotide sequences of
cAb-Lys2, cAb-Lys3, cAb-TT1 and cAb-TT2.
[0077] FIGS. 3A, 3B, 3C and 3D shown the immune response in
functional time for lysozyme carbonic anhydrase bovine erythrocytes
.alpha.-Amylase. See Example 12.
[0078] FIGS. 4A, 4B, 4C and 4D show the solid-phase binding of
fractionated IgG of D2/54 for RNaseA of the Amylase lysozyme and
carbonic anhydrase. See Example 13.
[0079] FIGS. 5A, 5B, 5C and 5D show the optical densities of bovine
and pancreatic .alpha.-Amylase for differing IgG's. See Example
14.
[0080] FIGS. 6A, 6B, 6C and 6D show optical densities for bovine
erythrocyte carbonic anhydrase for differing IgG's. Also see
Example 14.
[0081] FIG. 7 shows the chromatograph obtained in Example 16.
[0082] FIG. 8 shows a gene sequence. See Example 18.
[0083] FIGS. 9 and 10 show the gene sequences for respectably CA04
and CA05. See Example 19.
[0084] FIG. 11 shows a graph showing the affinity measurement of
CA04-HIS construct by competitive ELISA. See Example 20.
[0085] FIG. 12 shows a graph exhibiting the inhibition of carbonic
anhydrase. See Example 22.
[0086] FIG. 13 is a graph as explained in Example 7.
EXAMPLES
[0087] In the following the general strategy for obtaining
recognition molecules of the invention will be further illustrated
on the basis of specific recognition molecules against lysozyme and
tetanus toxin. The invention is however not limited to these target
molecules.
Example 1
[0088] Preparation of cAb-Lys2 and cAb-Lvs3
[0089] The procedure to obtain cAb-Lys2 and cAb-lys3 is disclosed
in the patent application WO 96 34103 published on Oct. 31,
1996.
Example 2
[0090] Introduction of Restriction Enzyme Sites in the Vicinity of
the N-part of the CDR3 Loop of cAb-Lys3
[0091] The pHEN4-.alpha.Lys3 (i.e. the plasmid of pHEN4 containing
the gene for camel VHH coding for the cAb-Lys3 protein) was taken
as a template and a PCR was performed with the VHBACK(A4) and the
SM020 primers. Another PCR is performed on the same template DNA
and with primers SM019 and AM006.
[0092] AM006 binds in the beginning of the gene pIII of pHEN4:
1 5'-CGTTAGTAAATGAATTTTCTGTATGAGG-3'
[0093] SM019 binds to codons 100 g to 100 m of cAb-Lys3 (Sal I site
underlined):
2 5'-CACGGTCTGTCGACGGGAGG-3'
[0094] SM020 binds to codons 100 m to 98 (Sal I site
underlined):
3 5'-CCTCCCGTCGACAGACCGTGGCC ACATTCATAATASNNAGCGTAG-3'
[0095] VHBACK(A4) binds in PelB leader signal of pHEN4 and
beginning of cAb-Lys3 codons 1 to 4 (Sfi I site is underlined):
4 5'-CATGCCATGACTCGCGGCCCAGCCGGCC ATGGCCGA(G/T)GT(G/C)CAGCT-3'
[0096] After digestion of both PCR fragments with Sal I, ligation
and final PCR with VHBACK(A4) and AM006 primers a DNA fragment is
generated which can be cloned in the pHEN4 cut with Sfi I and Bst
EII (The Bst EII site occurs naturally in framework 4 of all VHH
gene segments). The resulting phasmid DNA encodes a cAb-Lys 3 with
Ser100a randomized and in which codons
5 Leu100i.Ser100j.Thr100k CTT TCC ACT CTG TCG ACT
[0097] of the cAb-Lys3 are mutated. These silent mutations harbours
the restriction enzyme site for Sal I (underlined) and can be used
for subsequent cloning or clone characterization. Also the
nucleotides of the codons for
6 Cys100e.Gly100f.His100g TGT GGT CAC TGT GGC CAC,
[0098] are substituted. This silent mutation introduces a Bal I
site (underlined) which, like the Sal I site, can be used for
cloning or clone selection.
[0099] Using this strategy some 10,000 individual clones were
generated of which some 24 individual clones were toothpicked and
grown individually and tested separately for expression level and
binding to hen egg-white lysozyme. All clones contained a mutation
at position 100a and the sites for Sal I and Mlu I. Only two were
binding to the lysozyme although with slight a reduced affinity.
These mutations have the Ser100a substituted by respectively a Pro
and His. All other clones contained a different amino acid such as
Arg, Leu, or Lys, etc . . . and were shown to be expressed but
unable to bind the lysozyme to a reasonable extend.
[0100] This proves that it is possible to mutate the loop with
retention of the expression level but with an altered
affinity/specificity towards lysozyme compared to the original
cAb-Lys3.
Example 3
[0101] Complete Randomization of the `TUT` Loop
[0102] With the oligo's VHBACK(A4) and SM021 a DNA fragment was
generated by PCR on the cAb-Lys3 template which can be cloned after
Sfi I/Sal I digestion into the pHEN4-aLys3 mutant digested with the
same set of enzymes. The sequence of SM021 (Sal I site underlined)
binds to codons 100m to 100e and from 96 to 92:
7 5'-CCTCCCGTCGACAGACCGTGGCC ACA(SNN).sub.6CGAATCTGCCGCAC-3'
[0103] These plasmids have their codons coding for Thr97 up to
Glu100d removed and replaced by random codons (NNS).sub.6.
[0104] After construction of the library in a phage display vector
such as the pHEN4 the proper binders are selected by panning with
the elution/selection strategy explained in the description.
[0105] It is also possible to create a loop of different size.
Then, the sequence of the primer SM021 was changed to one in which
the random codons (NNS)X is varied with X=1 to 10 or more. The
smaller X, the smaller the loop, and the larger X, the more
extended the loop will be. These different libraries each with a
different loop size are used to find the best fit for the clefts of
the target proteins.
Example 4
[0106] Veneering of the `TUT` Loop of cAb-Lys3
[0107] A slight different methodology of the loop randomization
strategy generates a library in which the protruding loop from
cAb-Lys3 is changed only at its outer surface, while preserving
(most of) the internal and loop-structuring amino acids. This
strategy is referred to as `veneering`. The strategy consists of
changing the SM021 primer with an SM022 primer:
8 5'-CCTCCCGTCGACAGACCGTGGCCACASNNAT
A(SNN).sub.3GTA(SNN).sub.2CGAATCTGCCGCAC-3'
[0108] and to perform a PCR in combination with primer VHBACK(A4)
with pHEN4-.alpha.Lys3 as template. This primer anneals to the
codons 100 m to 92 and randomizes codons 97, 98, 100, 100a, 100b
and 100d, all others are retained. The randomized codons code for
amino acids which were found to be facing outward the loop. After
digestion of the PCR fragment with Sfi I and Sal I a library was
constructed into the pHEN4 vector digested with the same enzymes.
After expression of the mutated cAb on phage virions the proper
binders were selected by panning with the elution/selection
strategy explained in Example 3.
[0109] By changing the primer SM022 with a similar primer in which
the (SNN).sub.3 is exchanged by (SNN).sub.x (X=1 to 6 or more, but
not 3) and following the above protocol a library is generated in
which the tip of the protruding loop is shortened or extended
compared to the original loop. Also the other randomized positions
can be enlarged or shortened to create a `knob` on the side of the
protruding loop. These different libraries are used for panning
with the target molecules and selection of optimal binders.
Example 5
[0110] Modification of the Basic Recognition Unit Surrounding the
Protruding Loop
[0111] Once the recognition unit with the `TUT` motif is
constructed, the affinity or specificity can be increased by
(subtle) modifications of the basic recognition unit (or
`platform`) around the `TUT` loop. The three best sites for these
modifications are located at the N-terminal end of the recognition
unit, the loop around amino acids 72/75 and the CDR1 or CDR2
regions.
[0112] 1. The N-terminal end of the VHH is close to the antigen
binding loops. This site can be used as the site mentioned before
but the inserted amino acids will not be constrained. It is rather
a fusion product which will be obtained. Therefore this site is a
good site for inserting whole domains and for constructing
molecules with bispecificity.
[0113] 2. Reshaping the 72/75 loop for increasing the antigen
binding surface and the modulation of the affinity/specificity by
inserting/introducing new functional features. This loop is a good
site to introduce mutations (randomisations) as was observed in
camel and llama VHH clones insertions and deletions of one or two
amino acids at this position. This site can be extended by three
amino acids while the folding is still retained as well as its
antigen binding activity. The structure of the cAb-Lys3 in this
region is also not very well visible because of residual
flexibility in this region in the crystal. Therefore it is
anticipated that this region is a proper place to accommodate
deletion and insertions. The presence of the restriction enzyme
site Bsp HI around codons 81 to 82a
9 Leu.Met.Asn CTC.ATG.AAC
[0114] in combination with an oligonucleotide allows for
mutagenising this region with PCR based technology and standard
cloning techniques.
[0115] E.g. primer binding at codons 67 to 82b (Bsp HI site
underlined):
10 3'-AAGTGGTAGAGGGTT(XXX).sub.xTTG TGCCACATAGACGAGTACTTGTCG-5'
[0116] together with the VHBACK(A4) primer in a PCR reaction on
pHEN4-.alpha.Lys3 will generate a fragment which can be cloned into
pHEN4-.alpha.Lys3 digested with Bsp HI and Sfi I. The generated
library will encode the cAb-Lys3 protein in which the codons 72 to
75 are substituted by (XXX).sub.x. Depending on the size of nature
of the (XXX).sub.x codons between codons 72 and 75 can be
introduced, deleted or substituted.
[0117] For example, the introduction of Arg Gly Asp at this
location could turn the protein into an integrin, or the
randomization or inclusion of cysteine or histidines in this region
might allow the chelation of metals in this location to generate a
metallo-protein. In case one wants to incorporate a subdomain or a
domain of an enzyme at this position then it is suggested to
analyse the structure of the `new` protein to find the amino acids
enclosing the wanted domain with an orientation and distance
equivalent to the orientation and distance of amino acids 72 and 75
in the VHH. If the structure is unknown or no amino acid fulfills
this requirement, then it is advisable to introduce a short linker
peptide which will allow to span the difference so that no unwanted
constraints are imposed on the inserted domain which would inhibit
the folding, stability and function of the chimeric protein.
Similarly, changing the CDR1 or CDR2 amino acids can be performed
to increase the affinity and specificity of the recognition unit
with `TUT` motif.
Example 6
[0118] Increase of Lifetime of the Recognition Unit with `TUT`
Motif
[0119] The small single domain proteins of the invention have a
good tissue penetration, a good bio-distribution and a rapid
clearance from blood. For some applications (virus neutralization)
it is however beneficial to have a longer lifetime in blood. To
increase the lifetime within the blood, clone the protein with the
`TUT` motif can be cloned upstream of the hinge, CH2, and CH3
domains of IgG1 such as the IgG1 of human. This can be done by
standard cloning techniques. The BstEII site occurring in human and
cAb-Lys3 or other camel and llama VHH gene segments is a good site
for ligating the two gene fragments to each other. The pHEN4
expression vector can be used for bacterial expression of these
constructs, whereas the pCDNA-3 vector can be used for final
expression in mammalian cell lines. This is no problem as it is
reported that such constructs (without the CH1 and light chains)
are well expressed in both systems, and functional proteins are
obtained in these systems.
Example 7
[0120] Intrabodies
[0121] To interact with target proteins which are normally located
and/or functioning at internal cellular positions, it is necessary
to bring the small recognition unit with the `TUT` motif inside
this cellular compartment (cytosol, nucleus, endoplasmic reticulum,
mitochondria or chloroplast). The transformation of the
gene-segment of the small recognition unit with the `TUT` motif
behind a suitable promotor and/or localisation signal, or extended
with the SKDEL codons for targeting in the endoplasmic reticulum
allows for expression and direction of the designed molecule to the
cell compartment at will.
[0122] The cAb-TT2 was cloned behind the CMV promotor and a
chloroplast leader signal. Transformation of this construct in
Tobacco plants showed that the constructs were expressed and
functioning as measured by ELISA. 2 gram leafs of the transgenic
plant are grinded in a mortar in 2.5 ml PBS, 20% glycerol, 300
.mu.l of 10 mM PMSF (phenylsulfonylfluoride). After a desalting
over PD10 gelfiltration column (Pharmacia) we use 100 .mu.l
extract, 10 .mu.l extract+90 .mu.l water and twofold dilutions in
an ELISA. As control a non-transformed plant was used. Coating of
the microtiter wells is with 100 .mu.l 6.5 .mu.g/ml tetanus toxoid.
Blocking is with 1% casein in PBS, and detection of bound plant
cAb-TT2 is with rabbit anti-camel IgG and goat anti-rabbit alkaline
phosphatase conjugate (Sigma); paranitrophenylphosphate is the
substrate and reading is done at 405 nm after 15 minutes. Thus, our
small recognition units with `TUT` motif are useful for the
development as intrabodies.
Example 8
[0123] Peptido-mimics
[0124] Once a `TUT` loop is found and characterized, it is possible
to synthesise it chemically as a constrained peptide. This peptide
binds specifically and with good affinity inside the cavity of the
target protein as numerous contacts are made. The 11 amino acids
(from Asp95 to Cys100e) account for a surface of approximately 500
.ANG..sup.2 of contact with the lysozyme active site cleft. This
amount is certainly sufficient for generating specific binders from
oligopeptides. Thus, the synthesis of the oligopeptide such as
`CGDSTIYASYYEGS` (the underlined sequence is the protruding loop
part of cAb-Lys3, and the two Cys at the extremities serve to
constrain the peptide conformation by disulphide bond formation) is
used to test the specific binding to the lysozyme enzyme. In a
subsequent step it is possible by organic chemistry to synthesise
peptide analogues based on this peptide with similar binding
characteristics.
[0125] Characterised `TUT's` binding to other molecular clefts such
as those from enzymes or receptor molecules can be used to design
suitable peptido-mimics according to this strategy.
Example 8A
[0126] Production of Monoclonal Antibodies against cAb-TT2
[0127] The production of monoclonal antibodies (MAbs) was obtained
by an intrafootpad injection of the cAb-TT2 (5 .mu.gr) without tag
in complete Freunds adjuvant into a 4-6 weeks old female
BALB/c.times.C57/B/L,F1 mouse after eight days the mouse is
sacrified and the popliteal lymph nodes are removed. The cells are
mechanically released and washed once in DMEM medium.
[0128] The myeloma partner cells (NSO) are maintained in log-phase
growth in DMEM. The cells are washed once and counted. The lymph
node cells and the NSO cells are mixed in a 5 to 1 ratio and fused
with 50% polyethylene glycol (PEG 4000) in DMEM. The cells are
pelleted by centrifugation and resuspended gently in prewarmed
medium (DMEM-20% fetal calf serum containing hypoxanthine,
aminopterin, thymidine and streptomycin and penicillin as
antibiotics). The cells are transferred into 250 ml DMEM and
dispensed in microplates at approximately 5.times.10.sup.4 cells
per well. The cultures are incubated for ten days at 37.degree. C.,
5% CO.sub.2.
[0129] After ten days, the emergence of hybrid clones recorded. A
total of 227 colonies were observed and their supernatants are
tested in an ELISA. Purified cAb-TT2 at 1 .mu.g/ml in PBS) is
adsorbed overnight to wells of microtiter plates. The plates are
washed, blocked with 1% casein-PBS, and incubated with the culture
supernatants of the hybridoma cells for one hour at 37.degree. C.
The wells are washed again with PBS-tween20 (0,1%) and incubated
with goat anti-mouse immunoglobulin conjugated with alkaline
phosphatese (Sigma) for an additional hour. After the final wash,
the enzyme was detected with p-nitrophenyl phosphate dissolved in 1
M diethanolamine supplemented with 1 mM MgSO.sub.4 and adjusted
with HCl to pH 9.8. The color development is monitored at 405
nm.
[0130] Out of the 227 hybrid colonies about 10% (24) showed a
positive response in ELISA. From these 20 were selected for further
analysis. The isotype class and subclass typing showed that 20
clones were of the IgG isotypes (IgG1-type, IgG2a and IgG2b),
whereas the remaining four belong to the IgM class.
[0131] Testing the reactivity of 20 selected IgG monoclonals with
cAb-TT2 specificity against purified camel IgG1 (i.e. conventional
camel antibodies with light chains), IgG2 or IgG3 (i.e. camel
heavy-chain isotypes), three camel VHH with unknown specificities
(cAb-VHH9, cAb-VHH16 and cAb-VHH21), or cAb-lys1, cAb-TT1 and
cAb-TT2 indicated that strong responses are obtained with the
cAb-TT2, the original antigen for raising the monoclonals. Two
monoclonals (23G8 and 9A9) recognize weakly other VHH domains, as
well as camel IgG1. the supernatant of hybridoma 18C11, 21A3 and
3G2 mAbs give an intermediate response (50% of maximum response) to
camel IgG1 and mAb 15A11 recognizes two other non-related VHHs
(cAb-lys1 and cAb-VHH21) to a lower extend (20% of maximum
binding). Hence most of the anti-cAb-TT2 mAbs appear to be
monospecific for cAb-TT2 indicating a private idiotypic
specificty.
Example 9
[0132] Immunisations with the Recognition Units with `TUT`
Motif
[0133] The cAb-TT2 intra-footpad injection in BALB/c mouse led to
the generation of 24 hybridoma's (out of 227 hybrids tested) with
binding activity against cAb-TT2. From all these 24 monoclonals
only two could bind weakly to other camel VHH's, whereas the
binding to the cAb-TT2 was strong for all tested clones. Therefore,
the generated monoclonals are most likely anti-idiotypic
monoclonals.
[0134] It is known from the experiments of Zanetti (Nature 355, 476
(1992)) that the amino acids present at the CDR3 of the VH are
immunogenic and that it is possible to immunize a mouse against
malaria with a protein construct obtained by inserting a Plasmodium
epitope in the CDR3 of VH. In analogy, in the constructs mentioned
the protruding loop on the small recognition units is expected to
be a good site for inserting other loops for in vivo
immunizations.
[0135] It was realized that once a `TUT` against a particular
enzyme or receptor molecule is identified, it can be used to
generate monoclonal antibodies. It is expected that the
anti-idiotypic monoclonals will mimic the catalytic site of the
original enzyme or receptor. This strategy can be used to generate
Abzymes as the `TUT` replaces the `transition state of the
substrate` used to develop antibodies with catalytic activity. The
design and synthesis of stable `transition state of the substrate`
is often difficult or impossible. This strategy would bypass these
synthesis difficulties.
Example 10
[0136] Structure Analysis of cAb-Lys3 Co-crystallized with its
Antigen Lysozyme
[0137] This example is shown in FIG. 1 in the Article "Nature
Structural Biology" Volume 3, No. 9 from September 1996.
Example 11
[0138] Immunisation Protocols
[0139] Four different dromedaries (camelus dromedarius) are used
for immunisations with different antigens, or different amount of
antigen.
[0140] Dromedary 1
[0141] antigens:
[0142] Bovine RNase A at 0.1 mgr
[0143] Carbonic anhydrase at 0.1 mgr
[0144] b-lactamase at 1 mgr
[0145] Lysozyme at 1 mgr
[0146] Hepatitis B surface antigen serotype Ay at 0.25 mgr are
mixed in approximately 0.5 ml saline, together with an additional
two plant enzymes in a volume of 2 ml.
[0147] Day 0
[0148] Take 20 ml serum Inject antigen mixture emulsified with CFA
(equal volume), subcutaneous
[0149] Day 7
[0150] Take 20 ml serum
[0151] Day 14
[0152] Boost with antigen mixed in IFA, subcutaneous=tube DAY
14
[0153] Day 21
[0154] Take 20 ml serum
[0155] Day 28
[0156] Boost with antigen mixed in IFA, subcutaneous=DAY 28
[0157] Day 31
[0158] Take 20 ml blood (for lympocyte prep).
[0159] Day 35
[0160] Take 20 ml serum
[0161] Day 54
[0162] Boost with antigen mixed in IFA, subcutaneous=DAY 54
[0163] Day 57
[0164] Take 50 ml blood (for lymphocyte prep)
[0165] Day 61
[0166] Take 50 ml serum
[0167] Dromedary 2
[0168] antigens Bovine RNase A at 1 mgr
[0169] Carbonic anhydrase at 1 mgr Lysozyme at 1.4 mgr
.alpha.-amylase at 1 mgr
[0170] are mixed in approximately 0.25 ml saline.
[0171] Day 0
[0172] Take 20 ml serum Inject antigen mixed emulsified with CFA
(equal volume), subcutaneous
[0173] Day 7
[0174] Take 20 ml serum Boost with antigen mixed in IFA,
subcutaneous
[0175] Day 14
[0176] Take 20 ml serum Boost with antigen mixed in IFA,
subcutaneous
[0177] Day 21
[0178] Take 20 ml serum Boost with antigen mixed in IFA,
subcutaneous
[0179] Day 28
[0180] Take 20 ml serum Boost with antigen mixed in IFA,
subcutaneous
[0181] Day 31
[0182] Take 20 ml blood
[0183] Day 35
[0184] Take 20 ml serum Boost with antigen mixed in IFA,
subcutaneous
[0185] Day 42
[0186] Take 20 ml serum Boost with antigen mixed in IFA,
subcutaneous
[0187] Day 49
[0188] Take 20 ml serum Boost with antigen mixed in IFA,
subcutaneous
[0189] Day 54
[0190] Take 20 ml serum Boost with antigen mixed in IFA,
subcutaneous
[0191] Day 57
[0192] Take 50 ml blood
[0193] Day 61
[0194] Take 50 ml serum
[0195] Dromedary 3
[0196] antigens:
[0197] Bovine RNase A at 1 mgr
[0198] Carbonic anhydrase at 1 mgr
[0199] b-lactamase at 0.1 mgr
[0200] Lysozyme at 0.1 mgr
[0201] TAT at 0.5 mgr
[0202] Hepatitis B surface antigen serotype Ad at 0.25 mgr are
mixed in approximately 2.7 ml saline.
[0203] Day 0
[0204] Take 20 ml serum, take 20 ml blood (for preparation of
lymphocytes) Inject antigen mixture together with CFA (equal
volume), subcutaneous
[0205] Day 7
[0206] Take 20 ml serum
[0207] Day 14
[0208] Boost with antigen mixture in IFA, subcutaneous
[0209] Day 21
[0210] Take 20 ml serum
[0211] Day 28
[0212] Boost with antigen mixture in IFA, subcutaneous
[0213] Day 31
[0214] Take 20 ml blood (for lympocyte preparation)
[0215] Day 35
[0216] Take 20 ml serum
[0217] Day 54
[0218] Boost with antigen mixture in IFA, subcutaneous
[0219] Day 57
[0220] Take 50 ml blood (for lymphocyte preparation)
[0221] Dromedary 4
[0222] antigen: TAT
[0223] are mixed and used to generate neutralising antibodies. All
injections are done intramusculary.
[0224] Day 0
[0225] Take 20 ml serum, Inject 0.12 mgr cocktail+2 ml PBS+2 ml
IFA
[0226] Day 2
[0227] Priming with 0.24 mgr cocktail+2 ml PBS+2 IFA
[0228] Day 4
[0229] Priming with 0.36 mgr cocktail+2 ml PBS+2 IFA
[0230] Day 7
[0231] Priming with 0.48 mgr cocktail+2 ml 0.07 M sodiumphosphate+2
ml 0.07 M CaCl.sub.2
[0232] Day 9
[0233] Take 20 ml serum Priming with 0.96 mgr cocktail+2 ml 0,07 M
sodiumphosphate+2 ml 0.07 M CaCl.sub.2
[0234] Day 11
[0235] Priming with 1.50 mgr cocktail+2.5 ml 0.07 M
sodiumphosphate+2.5 ml CaCl.sub.2
[0236] Day 14
[0237] Priming with 1.98 mgr cocktail+3 ml sodiumphosphate+3 ml
CaCl.sub.2
[0238] Day 16
[0239] Priming with 1.32 mgr cocktail+2 ml sodiumphosphate+2 ml
CaCl.sub.2
[0240] Day 18
[0241] Priming with 1.74 mgr cocktail+2.5 ml sodiumphosphate+2.5 ml
CaCl.sub.2
[0242] Day 21
[0243] Priming with 2.16 mgr cocktail+3 ml sodiumphosphate+3 ml
CaCl.sub.2
[0244] Day 24
[0245] Priming with 1.80 mgr cocktail+3 ml sodiumphosphate+3 ml
CFA
[0246] Day 31
[0247] Take 20 ml blood and 20 ml serum
[0248] Day 65
[0249] Boost with 0.54 mgr cocktail+2 ml sodiumphosphate+2 ml
CaCl.sub.2
[0250] Day 72
[0251] Boost with 1.08 mgr cocktail+2 ml sodiumphosphate+2 ml
CaCl.sub.2
[0252] Day 75
[0253] Boost with 1.62 mgr cocktail+2.5 ml sodiumphosphate+2.5 ml
CaCl.sub.2
[0254] Day 79
[0255] Take 50 ml blood
[0256] Day 82
[0257] Take 50 ml serum
Example 12
[0258] Immune Response in Function of Time
[0259] Camel 2 (D2) has been injected with different antigens, as
described in example 11. Blood was collected and serum was removed
after coagulation.
[0260] Maxisorb plates were coated overnight at 4.degree. C.,
respectively with, as follows:
[0261] Lysozyme (3 .mu.g/ml in PBS)
[0262] Carbonic anhydrase bovine erythrocytes (4 .mu.g/ml in
PBS)
[0263] Pig pancreatic .alpha.-Amylase (3 .mu.g/ml in PBS)
[0264] RNase A
[0265] The procedure for immobilization of the enzyme included 30
minutes pretreatment of the Maxisorb plate with 0.25%
gluteraldehyde at room temperature. After washing with water,
RNaseA was then added at 10 .mu.g/ml in PBS and further incubated
overnight at 4.degree. C.
[0266] Plates were blocked for at least 2 hrs at room temperature
with 1% casein in PBS.
[0267] The sera were diluted in 0.1% casein/PBS. 100 .mu.l of the
diluted sera were added to the individual wells and incubated for 1
hr at room temperature.
[0268] Bound IgG's were detected with a rabbit polyclonal anti
camel IgG serum (I/1000 in 0.1% casein/PBS) followed by a
goat-anti-rabbit IgG Alkaline Phosphatase conjugate (1/1000
dilution in 0.1% casein/PBS). Between each step the wells were
washed 5.times. with 200 .mu.l PBS/0.1% Tw20.
[0269] 100 .mu.g p-nitro-phenyl-phosphate at 2 mg/ml in ELISA
buffer (10% diethanolamine buffer pH9.8 containg 0.5 mM
MgCl.sub.2,) was added and OD at 405 nm was measured after 20
minutes with Labsystems Multiscan RC ELISA plate reader. Optical
densities were not corrected for background.
Example 13
[0270] Solid-chase Binding of Fractionated 1eG of D2/54
[0271] 1. Fractionation of IgG
[0272] 1 ml of serum of camel day 54 was fractionated on
ProteinG/A. Protein concentrations were determined
spectrophotometrically at 278 nm, assuming a E.sub.1%=13.5.
[0273] 2. Coating of Maxisorb Plates
[0274] Coating was performed overnight in the cold room with
respectively:
[0275] Lysozyme Sigma L6876 (3 .mu.g/ml in PBS)
[0276] Carbonic anhydrase bovine erythrocytes Sigma C3934 (4
.mu.g/ml in PBS)
[0277] Pig pancreatic .alpha.-Amylase A6255 (3 .mu.g/ml in PBS)
[0278] RNase A.
[0279] The procedure for immobilization of the enzyme included 30
minutes pretreatment of the Maxisorb plate with 0.25%
gluteraldehyde. After washing with water, RNaseA was then added at
10 .mu.g/ml in PBS and further incubated overnight at 4.degree.
C.
[0280] Plates were blocked for at least 2 hrs at room temperature
with 1% casein in PBS.
[0281] 3. Detection of Bound IgG
[0282] Purified IgG's were individually tested on the individual
immobilized antigens in the range 5000-39 ng/ml. Dilution were made
in 0.1% casein/PBS.
[0283] 100 .mu.l of the diluted antibody solution was added to the
individual wells and after 1 hr incubation, bound IgG's were
detected with total rabbit serlun anti-camel IgG-home made and
subsequently with anti-rabbit-Alkaline Phosphatase conjugate (Sigma
no 8025). These reagents were diluted in 0.1% casein/PBS and were
used at a 1:1000 dilution. Between each step the wells were washed
5.times. with 200 .mu.l PBS/0.1% Tw20.
[0284] Finally 100 .mu.l p-nitro-phenyl-phosphate at 2 mg/ml in
ELISA buffer (10% diethanolamine buffer pH9.8 containg 0.5 mM
MgCl.sub.2) was added and OD at 405 nm was measured after 10
minutes with Labsystems Multiscan RC ELISA plate reader.
[0285] Optical densities were not corrected for background.
Example 14
[0286] Some Epitopes of Camel Heavy Chain IgGs are Cavities
[0287] In order to demonstrate that heavy-chain IgG with a long
CDR3-loop bind preferably to cavities, canyons or clefts present on
the surface of native protein, some binding experiments were
carried out.
[0288] As active sites of enzymes are preferably situated in the
largest cleft, the heavy-chain antibodies are especially suited for
development of inhibitors.
[0289] From binding experiments with as well .alpha.-amylase or
carbonic anhydrase in the presence or absence of competitive
inhibitors, it appeared that a substantial fraction of the heavy
chain IgGs bind to the active site.
[0290] 1. Bovine Pancratic .alpha.-amylase
[0291] Binding to solid phase enzyme of fractionated IgG1, IgG2a,
IgG2b and IgG3 (range 2500-19.5 ng:ml) in the presence or absence
of 1 mM Acarbose (pseudoheptasacharide with Ki 10.sup.-6M).
[0292] Bovine pancreatic .alpha.-amylase was coated overnight at
1.5 .mu.g/ml in PBS on Maxisorb plates at 4.degree. C. Plates were
blocked with 1% casein in PBS. Bound camel immunoglobulins were
detected with rabbit anti-camel antiserum (R17 1/1000 dilution),
followed by goat anti-rabit AP-conjugate (Sigma 1/1000 dilution).
Between each step the wells were washed 5.times. with 200 .mu.l
PBS/0.1% Tw20.
[0293] Finally 100 .mu.g p-nitro-phenyl-phosphate at 2 mg/ml in
ELISA buffer (10% diethanolamine buffer pH9.8 containg 0.5 mM
MgCl.sub.2) was added and OD at 405 nm was measured after 10
minutes with Labsystems Multiscan RC ELISA plate reader. Optical
densities were not corrected for background.
[0294] From these experiments it can be concluded that a
substantial portion of the amylase specific heavy chain antibodies
bind to or close to the active site of the enzyme. Even more
important, is the observation that the binding of IgG1 subclass to
the antigen, is not affected by the inhibitor.
[0295] 2. Bovine Erythrocyte Carbonic Anhydrase
[0296] Binding to solid phase enzyme of fractionated IgG1, IgG2a,
IgG2b and IgG3 in the presence or absence of 1 mM dorzolamide
(competitive inhibitor with Ki in the nanomolar range).
[0297] Carbonic anhydrase was coated overnight at 4 .mu.g/ml in PBS
on Maxisorb plates at 4.degree. C. Plates were blocked with 1%
casein in PBS. Bound camel immunoglobulins were detected with
rabbit anti-camel antiserum (R17 1/1000 dilution), followed by goat
anti-rabit AP-conjugate (Sigma 1/1000 dilution). Between each step
the wells were washed 5.times. with 200 .mu.l PBS/0.1% Tw20.
[0298] Finally 100 .mu.l p-nitro-phenyl-phosphate at 2 mg/ml in
ELISA buffer (10% diethanolamine buffer pH9.8 containg 0.5 mM
MgCl.sub.2) was added and OD at 405 nm was measured after 10
minutes with Labsystems Multiscan RC ELISA plate reader. Optical
densities were not corrected for background.
[0299] From these experiments it can be concluded that active site
binders for this enzyme are only present in the IgG3 subclass.
Example 15
[0300] Inhibition of Pancreatic Amylase by VHH of D2/61 IgG3
[0301] Based on the observation that the competitive inhibitor
acarbose was able to compete with the binding of heavy-chain
antibodies to solid-phase enzyme the following experiment was
carried out which demonstrates that part of these antibodies
inhibit the enzymatic activity of the enzyme. To rule out
immunoprecipitation as cause of reduced enzymatic activity, as to
expect the fractionated antibodies to be polyreactive and
polyclonal, VHH fragments of the IgG3 fraction were prepared.
[0302] These VHH from the IgG3 fraction of D2/61 were generated by
treatment with S.aureus V8 Endoglu-proteinase (Boehringer) in 0.1M
ammoniumbicarbonate pH8 (1/50 w/w enzyme/protein) for 2 hrs. The
efficiency of cleavage was followed by SDS-PAGE. After dialysis
against PBS non-digested material and Fc-fragments were removed by
protein G chromatography. The flow-through of the column contained
the VHH fragments (see example 16).
[0303] Residual enzymatic activity was determined using the
Ecoline.RTM. 25 Amylase assay (Merck-CNPG3 Method). The
ready-to-use substrate solution was diluted 10-fold with PBS to
lower the KSCN concentration to 90 mM in order to avoid chaotrope
induced dissociation.
[0304] Porcine pancreatic .alpha.-amylase (Sigma A-6255) was
diluted in 0.1% casein PBS to a concentation of 1.5 .mu.g/ml and 50
.mu.l of this solution was incubated with 100 .mu.l of the purified
VHH fragment (protein concentration 200 .mu.g/ml). After
preincubation for 60 minutes the enzymatic activity was determined
by adding part of the mixture to the 10-fold diluted substrate
solution. The enzymatic activity was calculated from the increase
in OD405 nm during 5 minutes. The enzymatic activity was reduced to
65%, relative to the enzymatic activity measured in the absence of
VHH fragments, thus demonstrating that inhibitory antibodies are
present.
Example 16
[0305] Digestion and Purification of VHH of IqG3 D2/61
[0306] VHH from the IgG3 fraction (1.72 mg/ml) of camel 2 bleeding
day 61 (D2/61) were generated by treatment with S.aureus V8
Endoglu-proteinase in 0.1M ammoniumbicarbonate pH8 (1/50 w/w
enzyme/protein ratio) for 2 hrs. The efficiency of cleavage was
followed by SDS-PAGE. After dialysis against PBS non-digested
material and Fc-fragments were removed by protein G chromatography.
The flow-through of the column contained the VHH fragments. The
protein concentration of the VHH top fraction VHH (200 .mu.g/ml)
was determined spectrofotometrically assuming a E1%=20. This
fraction was used for inhibition assays.
[0307] Digestion lgG3 fraction of D2/61 with V8 S.aureus protease
at pH8 in 0.1 M NH.sub.4HCO.sub.3 at 1/50 w/w enzyme/protein ratio
for 2 hrs.
[0308] 1. Molecular weight markers
[0309] 2. Undigested IgG3
[0310] 3. Digestion after 2 hr with Endo Glu-protease V8
[0311] 4-5-6. Frow-through of ProteinG-Sepharose column.
[0312] 7-8-9. Elution of ProteinG-Sepharose with Glycine/HCl pH
[0313] 2.7
Example 17
[0314] Preparation of Periferal Blood Lymphocytes
[0315] 4 Dromedaires are used. Approximately 7 ml of blood (in
EDTA) from each dromedary is collected and transported at 4.degree.
C. The blood is diluted with the same volume of sterile PBS and
layered on top of 50 ml tubes (Wak chemie). The tubes are spun at
1000 g for 20 minutes (2200 rpm) at 20.degree. C.
[0316] The liquid above the grid is transferred to a 50 ml Falcon
tube (To eliminate the blood platelets, it is better to remove the
supernatant and collecting only the lymphocytes which are banding
just above the grid).
[0317] The cells are spun down at 2500 rpm for 15 minutes at
4.degree. C. The pellet is resuspended in 0.5 ml PBS. After
dilution of a 10 .mu.l aliqout in 300 .mu.l PBS the cells are
counted. Each fraction contained approximately 3. 10.sup.7
cells/ml, of which only a minority were red blood cells. 5 tubes of
100 ml each were aliquotated in Eppendorf tubes and spun down at
2500 rpm, 5 min. The supernatant is removed and the pellet is
frozen at -80.degree. C. Each tube contains approximately 3.
10.sup.6 cells.
Example 18
[0318] VHH Library Construction from Peripheral Blood Lymphocytes
and Panning mRNA Preparation
[0319] The frozen lymphocytes (2 tubes, each 5.times.10.sup.6
lymphocytes/tube) collected from dromedary 2 (D2) at day 54, were
used to isolate mRNA with the Micro-FastTrack Kit (Invitrogen). The
mRNA was eleuted from the oligo-T solid support in 20 .mu.l water.
A total yield of 1.5 .mu.g mRNA was obtained as measured
spectrophotometrically (OD260 nm of 1 equals 35 .mu.gr
mRNA/m1).
[0320] cDNA Preparation
[0321] The cDNA was prepared from 1.5 .mu.gr mRNA with the cDNA
Cycle Kit (Invitrogen) according to the kit manufacturer
recommendations. The cDNA was purified by phenol/chloroform
extraction and by ethanol precipitation. The cDNA was resuspended
in a total volume of 100 .mu.l water.
[0322] PCR Amplification of VHH
[0323] The VHHs were amplified using 1 .mu.l of the cDNA sample
which is used as template in a PCR, using two gene specific primers
CH2FORTA4 and an equimolar mixture of primers SM017 and SM018, in a
total volume of 100 .mu.l with 2.5 units Taq Polymerase
(Boehringer) in the supplied buffer. Denaturation was at 94.degree.
C. for 1 minute, annealing at 55.degree. C. for 1 minute and
elongation at 72.degree. C. for 1 minute. This cycle was repeated
35 times.
11 CH2FORTA4: 5'-CGCCATCAAGGTACCAGTTGA-3' SM017:
5'-CCAGCCGGCCATGGCTGATGTGCAGCTGGTGGAGTCTGG-3' SM018:
5'-CCAGCCGGCCATGGCTCAGGTGCAGCTGGTGGAGTCTGG-3'
[0324] The most abondant amplification product had a size between
360 and 420 bp as visualised after gelelectrophoresis on 1.0%
agarose gel in TBE and 0.5 .mu.gr ethidium bromide/ml.
[0325] This PCR product was used as template for a reamplification
with nested PCR primers A4SHORT (containing a SfiI site,
underlined, the 15 nucleotides at its 3' end overlap with the 15
nucleotides at the 5' end of SM017 and SM018) and FRWRK4FOR (Not I
site underlined).
12 A4SHORT: 5'-CATGCCATGACTCGCGGCCCAGCCGGCCATGGC-3' FRWRK4FOR:
5'-GGACTAGTGCGGCCGCTGGAGACGGTGACCTGGGT-3'
[0326] The amplification product of 20 tubes was mixed and purified
by Geneclean (Bio 101, Inc.), and digested overnight at 37.degree.
C. with 50 units Not I and 50 units Sfi I (Gibco-BRL) in a total
volume of 200 .mu.l. The digested material was purified again by
Geneclean.
[0327] DHEN4 Vector Preparation.
[0328] The region around the multiple cloning site of pHEN1
phagemid vector (Hoogenboom et al., Nucleic acid Reseach, 19,
4133-4137, 1992) was modified, so that it now contained a SfiI and
NcoI site in the pe1B leader signal, and a Not I site preceding the
hemagglutinin tag of (Mullinax et al., Proc. Natl. Acad. Sci. USA
87, 8095-8099, 1990) (FIG. 8).
[0329] HindIII:1 SfiI:87 NcoI:98 PstI:115 BamHI:129 BstEII:135
NotI:149
[0330] Pe1B leader signal:40-105
[0331] HA-tag: 157-186
[0332] gen pIII: starts at 199
[0333] The phagemid (40 .mu.gr) was cleaved overnight with Sfi I
and Not I. The cloning vector was purified by agarose
gelelectrophoresis and Geneclean. The cut pHEN4 was eluted from the
glassmilk (Geneclean) with 40 .mu.l water.
[0334] VHH-vector Ligation.
[0335] The purified vector digested with Sfi and Not, and the
purified VHH Sfi-Not fragment were put on agarose gel to estimate
the concentration of the samples by ethidium bromide fluorescence.
Based on these estimations, 40 .mu.l (20 .mu.g) of vector and 40
.mu.l of VHH (5 .mu.g) were mixed (expected molar ratio of 1/4) and
ligated overnight at 16.degree. C. in a total volume of 100 .mu.l,
in 1.times. ligation buffer and 30 units T4 DNA ligase
(Boehringer). The DNA was thereafter purified by phenolization and
ethanol precipitation in the presence of 0.4 M LiCl. The DNA pellet
was washed with 70% ethanol, dried and finally resuspended 100
.mu.l water.
[0336] Electrocompetent Cells.
[0337] For the preparation of electrocompetent cells a preculture
of an isolated TG1 E.coli colony on minimal medium plate was
initiated. 1 ml of this preculture was transferred into 100 ml
2.times.TY medium supplemented with MgSO.sub.4 and grown at
18.degree. C. until an OD of 0.5 (600 nm) was reached. The cells
were harvested by centrifugation (3000 rpm, 10 min) and washed
several fold (at least 5 times) with water. The final cell pellet
was resuspended in 1 ml of 7% DMSO and aliquots of 50 .mu.l were
stored at -80.degree. C. until further use. A transformation
efficiency of more than 5.times.10.sup.8/.mu.gr pUC was
obtained.
[0338] Transformation, and Library Construction.
[0339] An aliquot of 1 .mu.l of the ligated DNA sample was added to
50 .mu.l electrocompetent TG1 cells in 2 mm electroporation
cuvettes (EUROGENTEC, Belgium) kept on ice. After
electrotransformation (2.5 kV, 25 MF, 200 Ohm), the cells are
immediately brought into 1 ml SOC medium and incubated at
37.degree. C. for 1 hour. Seventy of these tubes were mixed and
plated on a total of 50 large (24.3 cm.times.24.3 cm) LB agar
plates containing 100 .mu.gr ampicillin/ml to select for the
transformed cells and incubated overnight at 37.degree. C. At least
5.times.10.sup.6 individual transformants were obtained and these
were scraped from the plates with 2.times.TY medium, washed with
2.times.TY by centrifugation and finally resuspended in 100 ml
2.times.TY, 100 .mu.g/ml ampicillin, 1% glucose and 50% glycerol.
The bacterial suspension was frozen at -80.degree. C. until further
use.
[0340] M13K07 Helper Phase Preparation
[0341] A preculture of E.coli cells containing M13K07 is used to
inoculate 1 litre 2.times.TY medium, supplemented with 70 .mu.gr/ml
kanamycin, and is incubated overnight at 37.degree. C. with
vigourous shaking. The bacteria are removed by two centrifugations
(15 minutes, at 8000 rmp). The bacterial cells remaining in the
supernatant are heat inactivated by a 30 minutes incubation at
55.degree. C. The supernatant is filtrated through a 0.2.mu.
filter. The phages can be concentrated by PEG precipitation. To
this end 1/5 volume of 2.5 M Nacl, 20% PEG 8000 (200 ml) is added
to the 1 litre supernatant, and the mixture kept on ice for at
least 1 hour.
[0342] The sample is centrifugated for 40 minutes at 5000 rpm or
15-20 minutes at 13000 rpm. The M13K07 pellet is resuspended in
sterile PBS (10 ml). The concentration of the phages can be
determined spectrophotometrically (OD 1 at 260 nm corresponds to
4.times.10.sup.10 phages/ml), or the titer can be determined by
adding serial dilutions in 10 mM MgC12 to exponentially growing TG1
cells and plating the cells on LB plates containing 70 .mu.g/ml
kanamycin. (M13K07 carries the Kanamycin resistance gene). The
phages are brought to a titer of at least 10.sup.12 phages/ml.
[0343] Phase Rescue and Panning
[0344] 1. Phage Rescue
[0345] The cells transformed or carrying the pHEN4 recombinants are
grown in 2.times.TY, ampicillin (100 .mu.g/ml), 1% glucose. The
cells are pelleted once the culture reaches an OD of 0.6 (600 nM).
The cell pellet is washed in 2.times.TY medium and resuspended in
the same medium supplemented with ampicillin (100 .mu.gr/ml). The
cells are infected with M13K07 at a multiplicity of infection of 10
to 20. After an incubation period of 20 minutes at room
temperature, the cell suspension is bought to 70 .mu.gr
kanamycin/ml, and incubated overnight at 37.degree. C. with
vigourously shaking.
[0346] The virus particles and virions are purified by first
removing the bacterial cells through a centrifugation step (5000
rpm, 15 minutes) and filtration through a 0.4 or 0.2 .mu.m filter.
The phages are precipitates by addition of 1/4 volume of PEG
solution (20% PEG, 2.5M NaCl), and incubation on ice for at least
one hour. The phages are pelleted by 30 minutes centrifugation at
15,000 rpm. Occasionally an additional precipitation step was
included by resuspension of the phages in approximately 1 ml PBS
and adding 0.25 ml PEG solution. After incubation on ice for 30
minutes the phages can be pelleted by centrifugation 10 minutes,
13,000 rpm in an Eppendorf centrifuge. The phages are resuspended
in PBS (100 .mu.l). The concentration of the phages is measured by
UV absorption (260 nm), OD of 1 corresponds to a phage
concentration of 22.times.10.sup.10 phages/ml or a concentration of
44.times.10.sup.10 phagemid viri-ons/ml. The phages/phagemids are
brought to a concentration of 10.sup.12/ml with PBS, 0.1% casein
and used for panning.
[0347] 2. Panning
[0348] Two methods were used for panning. In one method Nunc
immunotubes (Nunc maxisop, startubes) were used to coat the
antigens overnight at 4.degree. C. (1 ml amylase (100 .mu.g/ml
PBS), or 1 ml carbonic anhydrase (100 .mu.g/ml PBS), 1 ml lysozyme
(200 .mu.g/ml PBS), or 1 ml RNase A (100 .mu.g/ml TBS/CaCl.sub.2 in
0.25% glutaraldehyde)). The tubes were washed 10 times with sterile
PBS, before incubation with the rescued virions. After one hour
incubation the non-bound virions and phages are removed by at least
10 washes with sterile PBS, Tween. The bound virions and phages are
eluted by adding 1 ml triethylamine (0.1 M), and incubation for 5
minutes at room temperature, neutralized with 0.5 ml 1 M Tris pH
7.4 2 ml of exponentially growing TG1 cells are added and after an
incubation period of 20 minutes the cells are plated on
LB/ampicillin plates. The next day the colonies are scraped from
the plates and can be used for the next round of panning after
rescue with M13K07.
[0349] For the second method 4 wells were used of a microtiter
plate for immobilizing the antigens (as above but with 100 .mu.l
volume/well). Washing of the wells, incubation with
phages/phagemids and elution, neutralization and TG1 infection is
as described above. Background is measured by adding virus
particles in wells which are only coated with the blocking agent
(1% Casein in PBS). The results for the four different antigens
were:
13 INPUT ELUTED BACKGROUND 1st round amylase 4 .times. 10.sup.10
0.06 .times. 10.sup.6 0.025 .times. 10.sup.6 carbonic anhydrase 4
.times. 10.sup.10 0.06 .times. 10.sup.6 0.025 .times. 10.sup.6
lysozyme 4 .times. 10.sup.10 0.1 .times. 10.sup.6 0.025 .times.
10.sup.6 RNase Ae 4 .times. 10.sup.10 0.06 .times. 10.sup.6 0.025
.times. 10.sup.6 2nd Round amylase 4 .times. 10.sup.11 1.3 .times.
10.sup.6 0.27 .times. 10.sup.6 carbonic anhydrase 4 .times.
10.sup.11 1.3 .times. 10.sup.6 0.056 .times. 10.sup.6 lysozyme 4
.times. 10.sup.11 0.26 .times. 10.sup.6 0.2 .times. 10.sup.6 RNase
A 4 .times. 10.sup.11 1.3 .times. 10.sup.6 0.048 .times. 10.sup.6
3rd Round amylase 4 .times. 10.sup.11 2.8 .times. 10.sup.6 0.08
.times. 10.sup.6 carbonic anhydrase 1 .times. 10.sup.8 0.05 .times.
10.sup.6 0.008 .times. 10.sup.6 lysozyme 1 .times. 10.sup.11 0.5
.times. 10.sup.6 0.016 .times. 10.sup.6 RNase A 1 .times. 10.sup.11
2.8 .times. 10.sup.6 0.004 .times. 10.sup.6
[0350] Selection of Individual Binders
[0351] After the last round of panning, the antigen binders are
selected by chosing randomly 24 individual clones from the plate
and growing the cells in 2.times.TY with 100 .mu.gr ampicillin/ml.
Two protocols were used to detect the presence of antigen binding
VHH. Either the VHH expression was induced with 1 mM IPTG when the
cells reached the exponential growing phase, or the cells were
infected with M13K07 helper phage. In the former strategy, the
antigen binding capacity of the cAb could be checked in an ELISA of
the culture supernatants with anti-HA-tag monoclonal (clone BBBB
BAbCo). In the second strategy the virions having antigen binders
on their tip are screened by ELISA with the anti-M13 detection kit
(Pharmacia).
[0352] In the ELISA experiment or the phage ELISA, we showed that
23 out of the 24 clones from the carbonic anhydrase pannings were
binding to the carbonic anhydrase. These clones were numbered CA01
till CA24. for the RNAseA pannings, all 24 clones scored positive,
these are referred to as RN01 till RN24. Plasmid DNA from clones
RN01 till RN12 was prepared and the insert was sequenced. RN02 and
RN06 are identical and RN06 was taken as reference. All other
clones are identical to the RN05 clone which was taken as reference
for the second set.
[0353] For the carbonic anhydrase 12 clones (CA01-CA12) were
sequenced. The sequence of CA01=CA06=CA07=CA09=CA12, the clones
CA04 and CA10 were unique and clones CA02, CA03, CA05, CA08 and
CA08 were identical with the exception of the presence of a silent
mutation in the CDR3 for CA05 and a different first amino acid
(which was forced by the PCR primer). So there occurred at least 4
different set of clones of which CA04, CA05, CA06, CA10 are taken
as the reference clones.
[0354] The nucleotide acid sequence of CA04 and CA05 is given in
example 19. It can be seen that both the CA04 and the CA05 clone
are indeed a VHH originating from a heavy chain antibody and not
from a conventional antibody (with light chain). The presence of
key markers Ser11 (codon 31-33nc), Phe37 (codon 109-111nc),
Glu44-Arg45 (codons 130-135nc) and Gly47 (codon 139-141nc) proves
this statement. The presence of a possible disulfide bridge between
CDR1-and CDR3 in both cases as indicated by the presence of
additional Cysteines (codons 97-99nc, and codon 313-315 for CA04 or
319-321nc for CA05) is also frequently observed in camel VHHs. The
long CDR3 of 18 amino acids for CA04 (codons 295-348nc) and of 19
amino acids (codons 289-345nc) for CA05 shows that both cAbs have a
long third hypervariable loop similar to that of cAb-lys3.
[0355] It will be shown in example 18a that CA04 binds into the
active site of the carbonic anhydrase, while CA05 does not. This
does not mean that the long loop of CA05 fails to bind into the
grooves of the antigen, as it is known from the crystal structure
of carbonic anhydrase that the active site for this enzyme is only
the second largest groove of the enzyme. The largest is located at
the other end from the active site, and it might be that the CA05
long CDR3 loop binds into this groove.
[0356] Three methods were used for panning. In the first method
Nunc immunotubes (ref) are used to coat the antigens (amylase,
carbonic anhydrase, lysozyme and RNase A). The tubes were washed 10
times with sterile PBS, before incubation with the rescued virions.
After a one hour incubation the non-bound virions and phages are
removed by at least 10 washes with sterile PBS, Tween. The bound
virions and phages are eluted by adding 1 ml triethylamine (0.1 M),
and incubating for 5 minutes at room temperature, neutralised with
0.5 ml 1 M Tris pH 7.4. 2 ml of exponentially growing TG1 cells are
added and after an incubation period of 20 minutes the cells are
plated on LB/ampicillin plates. The next day the colonies are
scraped from the plates and can be used for the next round of
panning afer rescue with M13K07.
[0357] For the second method 4 wells were used of a microtiter
plate for immobilising the antigens. Elution is with 100 .mu.l
triethylamine.
Example 18a
[0358] Binding of the Camel Single Domain Antibody CA04 into the
Active Site of Carbonic Anhydrase
[0359] All 24 clones isolated after panning with carbonic anhydrase
were induced with 1 mM IPTG. The expressed camel single domain VHHs
(cAbs) were extracted from the periplams and used in an ELISA
experiment in which the carbonic anhydrase was immobilised in the
wells of the microtiter plate. The periplasmic extracted proteins
(100 .mu.l) were incubated in the presence of 50 .mu.l PBS, or 50
.mu.l of a 2% solution dorzolamide (TRUSOPT.sup.R), or a 50 .mu.l
zcetazolamide soultion (DIAMOX.sup.R-Cyanamid). The two latter
drugs are binding into the active site of the carbonic anhydrase.
After 1 hour incubation, the wells are washed with PBS, Tween,
incubated with 1/5000 BABCO anti-HA antibody in 0.1% casein, PBS
for 1 hour at room temperature, washed and incubated with Rabbit
anti-mouse alkaline phosphatase conjugate (Sigma) at a 1/1000
dilution. Substrate is pare nitro phenyl phosphate (2 mg/ml) and
readings were done after 10 minutes at 405 nm. (table)
14 CLONES.backslash.INHIBITOR NONE Dorzolamide Acetazolamide CA01
0.75 0.143 0.17 CA02 1.04 0.85 0.90 CA03 1.04 0.86 0.94 CA04 0.74
0.22 0.26 CA05 0.85 0.75 0.77 CA06 0.62 0.25 0.27 CA07 0.87 0.23
0.28 CA08 1.22 1.06 1.120 CA09 0.83 0.17 0.23 CA10 0.68 0.64 0.64
CA11 1.00 0.93 0.92 CA12 0.79 0.14 0.18 CA13 0.89 0.15 0.19 CA14
0.68 0.13 0.30 CA15 0.22 0.12 0.14 CA16 0.88 0.46 0.47 CA17 0.48
0.12 0.13 CA18 0.73 0.13 0.17 CA19 0.74 0.13 0.17 CA20 0.74 0.13
0.17 CA21 0.84 0.15 0.20 CA22 0.84 0.15 0.19 CA23 1.04 0.99 1.01
CA24 1.13 1.09 1.15
[0360] Clone CA15 is not binding to carbonic anhydrase, or is a
weak binder.
[0361] Clone CA16 is only partially displaced by both dorzolamide
and acetazolamide.
[0362] Binding of cAb CA02, CA03, CA05, CA08, CA10, CA11, CA23 and
CA24 is not displaced by the active site binding drugs.
[0363] The cAbs of clones CA01, CA04, CA06, CA07, CA09, CA12, CA13,
CA14, CA17, CA18, CA19, CA20, CA21, CA22 are displaced by both the
dorzolamide and the acetazolamide. These cAbs are therefore
considered as active site binders. The ratio of active site binders
is 14 out of 24 clones. From the sequencing data of the CA01 to
CA12 we know that there are at least two different groups (CA04,
CA06) among the active site binders.
Example 19
[0364] Recloning and Expression of Binders with His6 Tag and
cAb-characterization
[0365] Recloning in pHEN6.
[0366] The HA tag and M13 pIII gene between the NotI and Eco RI
gene of pHEN4 was replaced by six His codons. Within the Sfi I and
Not I sites a cAb-Lys 3 gene was inserted (with the last Ser codon
`AGC` of the VHH replaced by `TCACGC", this will introduce an
additional Ser-Arg dipeptide). The following sequence is obtained
(figure) for pHEN6-Lys3.
[0367] The plasmid pHEN6-Lys3 is digested with HindIII and Bst EII
under optimal buffer and temperature conditions for the enzymes
(Gibco-BRL). The cAb-Lys3 containing fragment is further cleaved
with an additional digestion with NcoI. The linearised plasmid DNA
is purified by phenolisation and ethanol precipitation in the
presence of 0.4 M LiCl. The DNA is resuspended in 20 .mu.l water, 3
.mu.l is used to estimate the concentration by fluorescence in
agarose gel and the remaining material is brought to a
concentration of 100 ngr/.mu.l.
[0368] The pHEN4-CA04 or the pHEN4-CA05 are similarly digested by
HindIII and Not I. The cAb-CA04 and the cAb-CA05 containing
fragment are purified from agarose gel with Geneclean.
Approximately 100 ngr of these fragments (estimated from
fluorescence in agarose gel) are mixed with 100 ngr of HindIII-Not
I cut pHEN6 vector and ligated in a total volume of 10 .mu.l with
2.5 units T4 DNA ligase (Boehringer) overnight at room temperature.
The ligated DNA (2 .mu.l) is mixed with electrocompetent WK6 cells,
and plated on LB/ampicillin plates. The pHEN6-CA04 or pHEN6-CA05
containing colonies are screened by colony PCR with the universal
forward and reverse sequencing primer (standard PCR conditions).
Cutting the PCR fragment with Eco81I and separation of the
resulting fragments on 5% acrylamide gel allows the identification
and discrimination between residual pHEN6-Lys3 and pHEN6-CA04
orpHEN6-CA05 clones due to the larger CDR3 of the cAb-Lys3
insert.
[0369] The plasmids of the positively scored colonies were prepared
with alkaline lysis method and used as a template for
dideoxy-sequencing. The sequence of the pHEN6-CA04 and pHEN6-CA05
between the HindIII and Eco RI sites is given in the figures (the
cAb-CA04 and cAB-CA05 are in bold, and the his6-tag is
underlined).
[0370] Protein Expression and Purification
[0371] An overnight culture of WK6 cells freshly transformed with
plasmid pHEN6-CA04 and pHEN6-CA05 were used to inoculate 8 litre of
TB medium containing 100 .mu.gr/ml ampicillin and 0.1% glucose.
After growth at 37.degree. C. and when the culture reached an
absorbance of 0.75-1.0 at 600 nm, expression was induced by
addition of IPTG to a final concentration of 1 mM and cell growth
was continued for an additional 16 hours at 28.degree. C. The
periplasmic fractions were prepared essentially according to Skerra
and Pluckthun (Science 240, 1038-1041, 1988). Cells were harvested
by centrifugation at 4000 g for 10 minutes at 4.degree. C. and
resuspended in 1% of the original volume in icecold TES buffer (0.2
M Tris-HCl pH 8.0, 0.5 MM EDTA, 0.5 M sucrose). After one hour
incubation on ice, the cells were subjected to a mild osmotic shock
by the addition of 1.5% volume of ice-cold 1/4 diluted TES buffer.
After one hour incubation on ice, the cells were centrifugated
twice at 13000 g for 30 minutes at 4.degree. C. and PMSF (phenyl
methyl sulfonyl fluoride) to a final concentration of 1 mM was
added to the 200 ml of supematant which constituted the periplasmic
fraction.
[0372] This periplasmic fraction was concentrated 10 fold by
ultrafiltration in an Amicon cell (Millipore filter with MW cut off
of 5kDa) before being bound on a 2 Ni-NTA affinity column (Qiagen).
After washing with 40 of 50 mM sodiumphosphate buffer pH 8.0, 300
mM NACl, 10% glycerol buffer, the 6.times.His tagged single domain
antibody was eluted with a 40 ml linear gradient from 0 to 0.5 M
imidazole in the same buffer. The fractions containing cAb-CA04 or
cAb-CA05 respectively were pooled, concentrated 10 times by
ultrafiltration and the imidazole was removed by passing over a
Superdex-75 (Pharmacia) column using PBS buffer. 1.5 mgr pure
protein was obtained (as measured spectrophotometrically at 280 nm)
and concentrated by ultrafiltration to a concentration of 3
mg/ml.
Example 20
[0373] Affinity Measurement of CA04-HIS Constuct by Competitive
ELISA
[0374] Transformed TG1 cell were induced with IPTG for the
production of soluble protein. After harvesting the cells from 40
ml culture, the periplasmic fraction was prepared. In brief, the
pellet was resuspended in icecold TES (1.2 ml 50 mM TRIS pH 5 mM
EDTA, 20% sucrose) and incubated for 15 minutes on ice. After
centrifugation the supernatant was removed and the pellet was
resuspended in 1.2 ml of chilled water. The suspension was left on
ice for another 30 minutes. After centrifugation at 14.000 rpm the
supernatant was recovered and used in subsequent binding and
competition assays.
[0375] As well for binding as competition assays Carbonic Anhydrase
was coated on Maxisorb plates (Nunc) at a concentration of 1
.mu.g/ml in PBS (100 .mu.l overnight at 4.degree. C). Plates were
blocked with 200 .mu.l 1% casein in PBS) for 2 hr at room
temperature. For the competition assay, mixtures of the supernatant
at 1/100 dilution in 0.1% casein/PBS with free antigen varying in
concentration between 1-10.sup.4 nm were prepared. 100 .mu.l of
these mixtures were added to different wells of the plate. After 2
hr bound CA04-HIS was detected with Histidine tag specific
monoclonal antibody (Dianova, dia900, mouse monoclonal antibody
IgG1, anti (His).sub.6 tag) and subsequently with rabbit anti-mouse
alkaline phosphatase conjugate. Both secondary reagents were used
at a dilution 1/1000 in 0.1% casein/PBS. The substrate (100 .mu.l
of 2 mg/ml para-nitrophenolphosphate in ELISA buffer) was added and
OD 405 nm was measured after 15 minutes. From the plot of OD 405 nm
vs concentration of free antigen a Kd of 50 nM was estimated.
Example 21
[0376] Affinity Measurement and Kinetic Analysis of the
CA04:Carbonic Anhydrase Interaction
[0377] The kon, koff and Kd of the CA04 carbonic anhydrase
interaction were determined with an IAsys biosensor instrument.
[0378] An IAsys carboxymethyl dextraan cuvette (CMD) was used to
follow the interaction. The antigen was immobilized on the cuvette
by electrostatic absorption in the CMD matrix and by the subsequent
covalent reaction of lysyl groups with activated carboxyl groups on
the CMD polymer. Activation of the carboxyl groups was achieved by
the EDC/NHS coupeling chemistry (Johnson et al), using a EDC/NHS
coupeling kit (Affinity Sensors).
[0379] After a 7 min activation of the CMD cuvette, the cuvette was
washed with 10 mM NaAc buffer. 100 .mu.g/ml of carbonic anhydrase
was added to the cuvette and allowed to react for 10 minutes. After
washing the cuvette with PBS, the remaining activated carboxyl
groups were subsequently deactivated by adding 1M ethanolamine pH
8.0. After deactivation, several washes with 10 mm NaOH were
performed to remove all carbonic anhydrase which was not covalently
attatched. Calculation of the amount of immobilized antigen yielded
a value of 6 ng/mm. The stoichiometry of binding was measured by
adding a saturating amount of CA04 to the cuvette and was equal to
0.4.
[0380] All experiments were performed in PBS at 27.degree. C. and
at a stirr setting of 100. The regeneration conditions were
optimized. A one minute wash with 10 mM NaOH was used.
[0381] Binding traces for different concentrations of CA04 (2
10.sup.-8 to 1.5 10.sup.-7M) were performed in triplicate and
allowed to go to equilibrium. The curves were fitted with a single
exponential using FASTfit (Affinity Sensors). Baseline corrections
were taken into account. The resulting pseudo-first order rate
constants obtained from these fits were plotted against the
concentration of CA04. The kon was determined by linear regression
and yielded a value of 6.2 10.sup.5 M.sup.-1. The value is set as a
lower limit because of the occurance of mass transport limitations.
This was seen by plotting derivative of the signal versus the
signal for a high concentration of CA04 wich showed significant
curvature.
[0382] Dissociation phases, where after addition of saturating
amounts of CA04 the cuvette is washed with PBS, were followed in
the presence of 0.6 .mu.M of carbonic anhydrase (in triplicate).
The curves were fitted using the FASTfit software (Affinity
Sensors). The curves were fitted to a double exponential in wich
the slower phase was interpreted as being the result of rebinding
while the faster one reflects the actual off-rate. This value is
equal to 0.02s.sup.-1.
[0383] Calculation of the Kd based on the kinetic analysis yields a
value equal to 32 nM.
[0384] The Kd value was also determined by plottin the equilibrium
values versus the concentration of CA04 (3 10.sup.-8 to 1
10.sup.1-7M) and fitted to a hyperbolic relationship again using
FASTfit (Affinity Sensors). The Kd value obtained from this
analysis was equal to 60 nM.
Example 22
[0385] Inhibition of Bovine Erythrocyte Carbonic Anhydrase by
Ca04-His
[0386] Carbonic anhydrase (Sigma.C-3934) was dissolved in PBS and
the protein concentration was determined spectrophotometrically at
280 nm using a E1%=19.
[0387] The concentration of the purified CA04-His was determined
spectrophotometrically using a calculated extinction coefficient of
E1%=17 (PcGene). The enzyme was mixed at a fixed final
concentration of 2.3 .mu.M with variable amounts of CA04-His (range
1-8 .mu.M) in a constant volume of 60 .mu.l. After preincubation
for 15 minutes at room temperature, 945 .mu.l PBS and 5 .mu.l of
para-nitro-phenylacetate (2% solution in absolute ethanol) were
added (Pocker Y. and Stone J. T., Biochemistry, 6, 1967, 668-678).
The reaction mixture was transferred immediately to a cuvette and
the increase in OD405 nm was monitored for at least 5 minutes at
room temperature. The enzymatic velocities were corrected for
spontaneous hydrolysis of the substrate. Residual activity was
calculated relative to the enzymatic activity measured in the
absence of CA04-HIS.
Example 23
[0388] In vivo Neutralization of Tetanus Toxin
[0389] The in vivo neutralization test of tetanus toxin is
performed as described by Simpson et al., (J. Pharm. Exp.
Therapeutics 254, 98-103, 1990). Sixty-four NMRI mice (male and
female) of 8-12 weeks of age are randomly grouped in 8 groups (4
males and 4 females). The mice are injected i.p. with tetanus toxin
(RIT, Smith Kline Beecham, Rixensart, Belgium), antibody fragments
or both as follows:
[0390] group 1 PBS+cAb-TT1
[0391] group 2 PBS+cAb-TT2
[0392] group 3 PBS+Tetanus toxin (10.times.LD50)
[0393] group 4 PBS+Tetanus toxin (10.times.LD50)+cAb-TT1 (4
.mu.g)
[0394] group 5 PBS+Tetanus toxin (10.times.LD50)+cAb-TT1 (40
.mu.g)
[0395] group 6 PBS+Tetanus toxin (10.times.LD50)+cAb-TT2 (4
.mu.g)
[0396] group 7 PBS+Tetanus toxin (10.times.LD50)+cAb-TT2 (40
.mu.g)
[0397] group 8 PBS+Tetanus toxin (10.times.LD50)+non-specific
cAb-VHH21 (40 Ag)
[0398] The total volume of injection is 0.1 ml in all cases. The
mixture of VHHs and tetanus toxin is incubated for 30 minutes at
room temperature before injection. The mice are followed for two
weeks.
Sequence CWU 1
1
24 1 28 DNA Artificial Sequence oligonucleotide 1 cgttagtaaa
tgaattttct gtatgagg 28 2 20 DNA Artificial Sequence oligonucleotide
2 cacggtctgt cgacgggagg 20 3 43 DNA Artificial Sequence
misc_feature 36 random nucleotide 3 cctcccgtcg acagaccgtg
gccacattca taatanagcg tag 43 4 45 DNA Artificial Sequence
oligonucleotide 4 catgccatga ctcgcggccc agccggccat ggccgakgts cagct
45 5 46 DNA Artificial Sequence misc_feature 27..32 random
nucleotide 5 cctcccgtcg acagaccgtg gccacannnn nncgaatctg ccgcac 46
6 52 DNA Artificial Sequence misc_feature 27 random nucleotide 6
cctcccgtcg acagaccgtg gccacanata nnngtanncg aatctgccgc ac 52 7 44
DNA Artificial Sequence misc_feature (16)...(17) n can be 1-2
unknown nucleotides 7 aagtggtaga gggttnnttg tgccacatag acgagtactt
gtcg 44 8 14 PRT Camelus dromedarius 8 Cys Gly Asp Ser Thr Ile Tyr
Ala Ser Tyr Tyr Glu Gly Ser 1 5 10 9 21 DNA Artificial Sequence
oligonucleotide 9 cgccatcaag gtaccagttg a 21 10 39 DNA Artificial
Sequence oligonucleotide 10 ccagccggcc atggctgatg tgcagctggt
ggagtctgg 39 11 39 DNA Artificial Sequence oligonucleotide 11
ccagccggcc atggctcagg tgcagctggt ggagtctgg 39 12 33 DNA Artificial
Sequence oligonucleotide 12 catgccatga ctcgcggccc agccggccat ggc 33
13 35 DNA Artificial Sequence oligonucleotide 13 ggactagtgc
ggccgctgga gacggtgacc tgggt 35 14 357 DNA Camelus dromedarius CDS
(1)...(357) 14 gag gtg cag ctg cag gcg tct ggg gga ggc tcg gtg cag
gct gga ggg 48 Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Ser Val Gln
Ala Gly Gly 1 5 10 15 tct ctg aga ctc tcc tgt gcg gcc tct ggg gga
cag acc ttc gat agt 96 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly
Gln Thr Phe Asp Ser 20 25 30 tat gcc atg gcc tgg ttc cgc cag gct
cca ggg aag gag tgc gaa ttg 144 Tyr Ala Met Ala Trp Phe Arg Gln Ala
Pro Gly Lys Glu Cys Glu Leu 35 40 45 gtc tcg agt att att ggt gat
gat aac aga aac tat gcc gac tcc gtg 192 Val Ser Ser Ile Ile Gly Asp
Asp Asn Arg Asn Tyr Ala Asp Ser Val 50 55 60 aaa ggc cga ttc acc
atc tcc cga gac aac gcc aag aac acg gta tat 240 Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 ctg caa atg
gac cgt ctg aat cct gag gac acg gcc gtg tat tac tgt 288 Leu Gln Met
Asp Arg Leu Asn Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gcg
caa ttg ggt agt gcc cgg tcg gct atg tac tgt gcg ggc cag ggg 336 Ala
Gln Leu Gly Ser Ala Arg Ser Ala Met Tyr Cys Ala Gly Gln Gly 100 105
110 acc cag gtc acc gtc tcc tca 357 Thr Gln Val Thr Val Ser Ser 115
15 119 PRT Camelus dromedarius 15 Glu Val Gln Leu Gln Ala Ser Gly
Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Gly Gln Thr Phe Asp Ser 20 25 30 Tyr Ala Met Ala
Trp Phe Arg Gln Ala Pro Gly Lys Glu Cys Glu Leu 35 40 45 Val Ser
Ser Ile Ile Gly Asp Asp Asn Arg Asn Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65
70 75 80 Leu Gln Met Asp Arg Leu Asn Pro Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Gln Leu Gly Ser Ala Arg Ser Ala Met Tyr Cys
Ala Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115 16 381
DNA Camelus dromedarius CDS (1)...(381) 16 gag gtg cag ctg cag gcg
tct gga gga ggc tcg gtg cag gct gga ggg 48 Glu Val Gln Leu Gln Ala
Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 tct ctg agg ctc
tct tgt aca gcc gct aat tac gcc ttt gat tcc aag 96 Ser Leu Arg Leu
Ser Cys Thr Ala Ala Asn Tyr Ala Phe Asp Ser Lys 20 25 30 acc gtg
ggc tgg ttc cgc cag gtt cca gga aag gag cgc gag ggg gtc 144 Thr Val
Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val 35 40 45
gcg ggt atc agt agt ggt ggc agt acc aca gcc tat tcc gac tcc gtg 192
Ala Gly Ile Ser Ser Gly Gly Ser Thr Thr Ala Tyr Ser Asp Ser Val 50
55 60 aag ggc cga tac acc gtc tcc ctt gag aac gcc aag aac act gtg
tat 240 Lys Gly Arg Tyr Thr Val Ser Leu Glu Asn Ala Lys Asn Thr Val
Tyr 65 70 75 80 cta ctg ata gac aac cta caa cct gaa gac act gcc ata
tac tac tgc 288 Leu Leu Ile Asp Asn Leu Gln Pro Glu Asp Thr Ala Ile
Tyr Tyr Cys 85 90 95 gca gga gtg agc ggt tgg cga ggg cgg cag tgg
ctg cta ctg gca gag 336 Ala Gly Val Ser Gly Trp Arg Gly Arg Gln Trp
Leu Leu Leu Ala Glu 100 105 110 acc tat cgg ttc tgg ggc cag ggg act
cag gtc acc gtc tcc tca 381 Thr Tyr Arg Phe Trp Gly Gln Gly Thr Gln
Val Thr Val Ser Ser 115 120 125 17 127 PRT Camelus dromedarius 17
Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ala Asn Tyr Ala Phe Asp Ser
Lys 20 25 30 Thr Val Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg
Glu Gly Val 35 40 45 Ala Gly Ile Ser Ser Gly Gly Ser Thr Thr Ala
Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Tyr Thr Val Ser Leu Glu
Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Leu Ile Asp Asn Leu Gln
Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Gly Val Ser Gly
Trp Arg Gly Arg Gln Trp Leu Leu Leu Ala Glu 100 105 110 Thr Tyr Arg
Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 18 384
DNA Camelus dromedarius CDS (1)...(384) 18 gag gtc cag ctg cag gcg
tct gga gga ggc tcg gtg cag gct gga cag 48 Glu Val Gln Leu Gln Ala
Ser Gly Gly Gly Ser Val Gln Ala Gly Gln 1 5 10 15 tct ctg aga ctc
tcc tgt gcg acc tct gga gcc acc tcc agt agc aac 96 Ser Leu Arg Leu
Ser Cys Ala Thr Ser Gly Ala Thr Ser Ser Ser Asn 20 25 30 tgc atg
ggc tgg ttc cgc cag gct cca ggg aag gag cgc gag ggg gtc 144 Cys Met
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45
gca gtt att gat act ggt aga ggg aat aca gcc tat gcc gac tcc gtg 192
Ala Val Ile Asp Thr Gly Arg Gly Asn Thr Ala Tyr Ala Asp Ser Val 50
55 60 cag ggc cga ttg acc atc tcc tta gac aac gcc aag aac acg cta
tat 240 Gln Gly Arg Leu Thr Ile Ser Leu Asp Asn Ala Lys Asn Thr Leu
Tyr 65 70 75 80 ctg caa atg aac agc ctg aaa cct gag gac act gcc atg
tac tac tgt 288 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met
Tyr Tyr Cys 85 90 95 gca gca gat aca tcc act tgg tat cgt ggt tac
tgc gga aca aat cca 336 Ala Ala Asp Thr Ser Thr Trp Tyr Arg Gly Tyr
Cys Gly Thr Asn Pro 100 105 110 aat tac ttt tcg tac tgg ggc cag ggg
acc cag gtc acc gtc tcc tca 384 Asn Tyr Phe Ser Tyr Trp Gly Gln Gly
Thr Gln Val Thr Val Ser Ser 115 120 125 19 128 PRT Camelus
dromedarius 19 Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Ser Val Gln
Ala Gly Gln 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Ala
Thr Ser Ser Ser Asn 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro
Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Val Ile Asp Thr Gly Arg
Gly Asn Thr Ala Tyr Ala Asp Ser Val 50 55 60 Gln Gly Arg Leu Thr
Ile Ser Leu Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met
Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala
Ala Asp Thr Ser Thr Trp Tyr Arg Gly Tyr Cys Gly Thr Asn Pro 100 105
110 Asn Tyr Phe Ser Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125 20 399 DNA Camelus dromedarius CDS (1)...(399) 20 gat
gtg cag ctg cag gcg tct gga gga ggc tcg gtg cag gct gga ggg 48 Asp
Val Gln Leu Gln Ala Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10
15 tct ctg aga ctc tcc tgt gca gcc tct gga tac acc atc ggt ccc tac
96 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ile Gly Pro Tyr
20 25 30 tgt atg ggg tgg ttc cgc cag gcc cca ggg aag gag cgt gag
ggg gtc 144 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
Gly Val 35 40 45 gca gca att aat atg ggt ggt ggt atc acc tac tac
gcc gac tcc gtg 192 Ala Ala Ile Asn Met Gly Gly Gly Ile Thr Tyr Tyr
Ala Asp Ser Val 50 55 60 aag ggc cga ttc acc atc tcc caa gac aac
gcc aag aac acg gtg tat 240 Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn
Ala Lys Asn Thr Val Tyr 65 70 75 80 ctg ctc atg aac agc cta gaa cct
gag gac acg gcc atc tat tac tgt 288 Leu Leu Met Asn Ser Leu Glu Pro
Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 gcg gca gat tcg acc atc
tac gct agt tat tat gaa tgt ggt cac ggt 336 Ala Ala Asp Ser Thr Ile
Tyr Ala Ser Tyr Tyr Glu Cys Gly His Gly 100 105 110 ctt tcc acg gga
gga tat ggg tat gac tcc tgg ggc cag ggg acc cag 384 Leu Ser Thr Gly
Gly Tyr Gly Tyr Asp Ser Trp Gly Gln Gly Thr Gln 115 120 125 gtc acc
gtc tcc tca 399 Val Thr Val Ser Ser 130 21 133 PRT Camelus
dromedarius 21 Asp Val Gln Leu Gln Ala Ser Gly Gly Gly Ser Val Gln
Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr
Thr Ile Gly Pro Tyr 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro
Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Asn Met Gly Gly
Gly Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr
Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Leu Met
Asn Ser Leu Glu Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala
Ala Asp Ser Thr Ile Tyr Ala Ser Tyr Tyr Glu Cys Gly His Gly 100 105
110 Leu Ser Thr Gly Gly Tyr Gly Tyr Asp Ser Trp Gly Gln Gly Thr Gln
115 120 125 Val Thr Val Ser Ser 130 22 211 DNA Camelus dromedarius
22 aagcttgcat gcaaattcta tttcaaggag acagtcataa tgaaatacct
attgcctacg 60 gcagccgctg gattgttatt actcgcggcc cagccggcca
tggcccaggt gcagctgcag 120 gagctcgagg atccggtcac cgtctccagc
ggccgctacc cgtacgacgt tccggactac 180 ggttccggcc gagcatagac
tgttgaaagt t 211 23 524 DNA Camelus dromedarius 23 aagcttgcat
gcaaattcta tttcaaggag acagtcataa tgaaatacct attgcctacg 60
gcagccgctg gattgttatt actcgcggcc cagccggcca tggctcaggt gcagctggtg
120 gagtctgggg gaggctcggt gcagactgga gggtctctga gactctcctg
tgcagcctct 180 ggatacacct acactaggcg ctgcatggcc tggttccgcc
aggctccagg aaaggagcgc 240 gagggggtcg cacttattta tattgatggt
ggtaggacag actatgccga ctccgcgaag 300 ggccgattca ccatctccca
agaccgcgcc aagaacacgg tgtatctgca aatgaacagc 360 ctgaaacctg
aggacactgc catgtactat tgtgcaggag atgggggcag attagatcct 420
tactgctcaa ttaaggcata tgcgtatagg tactggggcc aggggaccca ggtcaccgtc
480 tcctcacgcg gccgccacca ccatcaccat cactaataga attc 524 24 521 DNA
Camelus dromedarius 24 aagcttgcat gcaaattcta tttcaaggag acagtcataa
tgaaatacct attgcctacg 60 gcagccgctg gattgttatt actcgcggcc
cagccggcca tggctcaggt gcagctggtg 120 gagtctgggg gaggctcggt
gcaggctgga gggtctctga gactctcctg tgcagcctct 180 ggatacaccg
tcagtaccta ctgcatgggc tggttccgcc aggctccagg gaaggagcgt 240
gagggggtcg caactattct cggtggtagc acatactacg gcgactccgt gaagggccga
300 ttcaccatct ctcaagacaa cgccaagaac acggtgtatc tgcaaatgaa
cagcctgaaa 360 cctgaggata cggccatcta ttattgtgcg ggatcgacgg
tggccagtac tggttggtgc 420 tcccgtctaa ggccgtatga ctaccactat
cggggccagg ggacccaggt caccgtctcc 480 tcacgcggcc gccaccacca
tcaccatcac taatagaatt c 521
* * * * *